Compositions and methods for treating pulmonary edema or lung inflammation

ABSTRACT

A pharmaceutical composition for administering directly to the pulmonary tract of a subject includes a salt of triiodothyronine and a pharmaceutically acceptable buffer, adjusted to pH of 5.5-8.5. The composition can be administered prophylactically or therapeutically to a subject to treat lung inflammation or pulmonary edema.

CROSS-REFERENCE TO RELATED APPLICATION

This application is the § 371 U.S. National Stage of InternationalApplication No. PCT/US2019/016068, filed Jan. 31, 2019, which claimspriority to U.S. Provisional Patent Application No. 62/624,631 filedJan. 31, 2018, each of which is incorporated herein by reference in itsentirety.

GOVERNMENT FUNDING

This invention was made with government supportt under HL050152 awardedby the National Institutes of Health. The government has certain rightsin the invention.

SUMMARY

This disclosure describes, in one aspect, a pharmaceutical compositionfor administering directly to the pulmonary tract (e.g., nasosinus,intratracheal, intrabronchial, or alveolar airspace) of a subject.Generally, the composition includes a salt of triiodothyronine and apharmaceutically acceptable buffer, adjusted to a pH of 5.5-8.5.

In some embodiments, the salt of triiodothyronine is provided in anamount of at least 5 μg per 10 ml.

In some embodiments, the composition is aerosolized. In otherembodiments, the composition is nebulized.

In another aspect, this disclosure describes a method for treating asubject having, or at risk of having inflammation of lung tissues.Generally, the method includes administering to the subject an amount oftriiodothyronine effective to ameliorate lung inflammation, wherein thetriiodothyronine is administered directly to the pulmonary tract.

In some embodiments of either method, the triiodothyronine isadministered by instillation. In other embodiments of either method, thetriiodothyronine is administered by inhalation of an aerosolizedformulation. In still other embodiments of either method, thetriiodothyronine is administered by inhalation of a nebulizedformulation.

In some embodiments of either method, the triiodothyronine isadministered to a lung-delivered drug dose range of 10 ng to 5 mg.

In some embodiments of either method, the triiodothyronine isadministered prior to the subject manifesting any symptom or clinicalsign of lung inflammation or pulmonary edema.

In some embodiments of either method, the triiodothyronine isadministered after the subject manifests a symptom or clinical sign oflung inflammation or pulmonary edema.

In some embodiments of either method, the lung inflammation or pulmonaryedema is a clinical sign of acute respiratory distress syndrome (ARDS).

In some embodiments of either method, the lung inflammation or pulmonaryedema is a symptom or clinical sign of premature birth, chest trauma,congestive heart failure, lung transplant, lung cancer radiotherapy,lung cancer chemotherapy, smoking, exposure to a pollutant,hypersensitivity pneumonitis, a reactive/obstructive lung disease,aspiration chemical pneumonitis/pneumonia, pneumonia, an infection ofthe nasosinus, intratracheal, intrabronchial or alveolar airspace, aconnective tissue disease, Wegener's granulomatosis, Good pasturedisease, acute eosinophilic pneumonia, chronic eosinophilic pneumonia,medication-related lung injury, cryptogenic organizing pneumonia,Churg-Strauss syndrome , congenital lung disease, or structural lungdisease.

The above summary is not intended to describe each disclosed embodimentor every implementation of the present invention. The description thatfollows more particularly exemplifies illustrative embodiments. Inseveral places throughout the application, guidance is provided throughlists of examples, which examples can be used in various combinations.In each instance, the recited list serves only as a representative groupand should not be interpreted as an exclusive list.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing orphotograph executed in color. Copies of this patent or patentapplication publication with color drawings or photographs will beprovided by the Office upon request and payment of the necessary fee.

FIG. 1. Immunohistochemical localization of the enzyme deiodinase-3 (D3)in lung tissue from acute respiratory distress syndrome (ARDS) patientsand normal human. (A) ARDS tissue samples showed characteristic diffusealveolar damage and proteinaceous alveolar filling in the air spaceswith diffusely positive D3 staining in multiple cell types. (D) ARDStissue samples showed hyaline membrane formation with D3-positivestaining of type II alveolar pneumocytes. (G) ARDS tissue samples showedproliferation and inflammatory cells in the interstitium withD3-positive staining of spindle-shaped cells and capillary endothelium.(B, E, H) Control tissue samples from normal human lung demonstratednormal histologic architecture of the alveoli and interstitium withoutsignificant D3 staining. (C, F, I) ARDS samples processed with normalmouse serum, rather than primary antibody (as a specificity control),also demonstrated no significant D3 staining.

FIG. 2. Deiodinase-3 activity and T3 quantity in lung tissue of ARDS andnormal human lungs. Lung D3 enzymatic activity is elevated in early ARDSlungs and lung T3 concentration is decreased in both early and late ARDSlungs. D3 activity (A) and total T3 concentrations (B) were measured inpost-mortem human early ARDS (n=3), late ARDS (n=5) and normal control(n=4) lungs. Data are presented as mean±SEM. Groups not sharing a commonsuperscript are significantly different by one-way ANOVA and Tukey'smultiple comparison test (P<0.05).

FIG. 3. Free T3 levels after single or dual T3 dosing protocols. Mean±SDfree triiodothyronine (T3) levels by dosing groups for human patientsreceiving only bolus of triiodothyronine as follows: 0.05 μg/kg at 0hours+0.1 μg/kg at three hours (open triangles), 0.2 μg/kg at 0 hours(filled circles), or 0.4 μg/kg at 0 hours (filled diamonds). The shadedbox represents the normal range for serum free triiodothyronine levels.

FIG. 4. Design of FDA-approved Phase I/II clinical human trial of T3instillation for treating ARDS.

FIG. 5. Time course of change of serum T3 concentration (mean+/−SEM) vs.time in a rat model. A single dose of T3 was administered viaintratracheal instillation at a dose of 2.7 (˜10.0 μg/kg). Samples wereanalyzed for total T3 using a chemiluminescence assay. Wheneverpossible, mean concentrations were derived from threeanimals/gender/time point.

FIG. 6. Serum T3 concentration (mean+/−SEM) following intravenous orintratracheal administration of liothyronine sodium (2.7 μg in 300 μl,pH 7.5) to rats. A single dose of T3 was administered intravenously(diamonds) or via intratracheal instillation (squares). Samples wereanalyzed for total T3 using a chemiluminescence assay.

FIG. 7. T3 increases RLE-6TN cell survival under 95% oxygen and duringrecovery in normoxia. (A) T3 increases RLE-6TN cell survival underhyperoxia stress. The cells were incubated in 90% O₂ and 5% CO₂ for 72hours in the present of T3 (10⁻⁶M for A) or RT3 (10⁻⁶M for A) in 2%stripped FBS culture medium. (B) T3 Dose curve for RLE-6TN cell survivalunder hyperoxia. The cells were cultured in DMEM/F12 with 10% FBS in 21%O₂ and 5% CO₂ overnight. The cells were then incubated in 90% O₂ and 5%CO₂ for 72 hours in the presence of indicated concentrations of T3 inDMEM/F12 medium supplemented with 2% stripped FBS. (C) RLE-6TN cellswere incubated with/without T3 in hyperoxia for 72 hours, and cells werethen transferred to room air and cultured in DMEM/F12 with 10% FBS foranother 72 hours. The viable cells are counted immediately after 72-hourhyperoxia exposure. Cell viability is assessed with trypan blue. Theamount of viable cells under specific conditions is presented as apercentage of the cell number from hyperoxia alone. Data are mean±s.d.of four independent experiments with *=P<0.05; **=P<0.01.

FIG. 8. T3 increases the number of RLE-6TN cells after hyperoxia injury.(A) RLE-6TN cells were exposed to hyperoxia for 24 hours, then weretransferred to room air for 48 hours in the presence of T3 or rT3 inDMEM/F12 medium supplemented with 2% stripped FBS. (B) RLE-6TN cellswere exposed to hyperoxia for 48 hours, then were transferred to roomair for 48 hours in the presence of T3 or rT3 in DMEM/F12 mediumsupplemented with 2% stripped FBS. In both (A) and (B), the number ofviable cells under specific conditions is presented as a percentage ofthe cell number related to hyperoxia alone in the same experiment. Dataare mean±s.d. of four independent experiments with *=P<0.05; **=P<0.01.

FIG. 9. Protective effect of T3 on hyperoxic damage of ATII requires theNrf2 activation. RLE-6TN cells were incubated with or without 10⁻⁶ M T3in 90% O₂ and 5% CO₂ for 24 hours in the presence of T3 (10-6M) inDMEM/F12 medium supplemented with 2% stripped FBS. The cells were thencollected for Western Blotting analysis. (A) Cellular total protein ofNrf2. (B) Nuclear Nrf2.

FIG. 10. HO-1 Upregulation is required for T3-increased RLE-6TN cellsurvival in hyperoxia. (A) T3 increases total cellular HO-1 proteinunder hyperoxia. The cells were incubated in 90% O₂ and 5% CO₂ for 72hours in the present of T3 (10⁻⁶M). The cells were then collected forWestern Blot analysis. (B) The cells were incubated in 21% O₂ and 5% CO₂for 72 hours in the present of HO-1 inhibitor Tin ProtoporphyrinIX(10-6M) tinT3 (10⁻⁶M). (C) The cells were incubated in 90% O₂ and 5%CO₂ for 72 hours in the present of T3 (10⁻⁶M) or Tin Protoporphyrin(10-6M). Cell viability is assessed with trypan blue. The number ofviable cells under specific conditions is presented as a percentage ofthe cell number related to hyperoxia alone. Data are mean±s.d. of threeindependent experiments with *=P<0.05; **=P<0.01.

FIG. 11. PI3K inhibitor wortmannin blocked T3-induced cell survival inhyperoxia. The cells were incubated in 90% O₂ and 5% CO₂ for 72 hours inthe present of T3 (10⁻⁶M) or wortmannin (10⁻⁶M) in DMEM/F12 mediumsupplemented with 2% stripped FBS. Cell viability is assessed withtrypan blue. The number of viable cells under specific conditions ispresented as a percentage of the cell number related to hyperoxia alone.Data are mean±s.d. of four independent experiments with *=P<0.05;**=P<0.01.

FIG. 12. T3-induced increase in Nrf2 and HO-1 protein via PI3K inhyperoxia. (A) Western Blot and densitometry data for total Nrf2. (B)Western Blot and densitometry data for cytoplasmic Nrf2. (C) WesternBlot and densitometry data for nuclear Nrf2.

FIG. 13. Hyperoxia decreased serum total T3 concentration. Rats wereexposed to ˜95% of oxygen for 60 hours. T3 supplementation started after24 hours of hyperoxia exposure. Data are presented as mean±standarddeviation (SD) of independent experiments (8 rats for room air control,10 rats for hyperoxia, six rats for T3 supplementation) ***, p<0.001.RA: room air control.

FIG. 14. Effects of T3 on hyperoxia-induced indicia. (A) T3 diminisheshyperoxia-induced increases of wet/dry lung weight ratio. (B) T3diminishes hyperoxia-induced increases of bronchoalveolar lavage (BAL)fluid protein concentration. Data are presented as mean±SD ofindependent experiments (four rats from two experiments for 48-hourexposure; three rats from three experiments for 60-hour exposure). *,p<0.05; **, p<0.01. RA: room air control.

FIG. 15. T3 decreases hyperoxia-induced increase of BALF nucleatedcells. Data are presented as mean±SD of independent experiments (fourrats from two experiments for 48-hour exposure; four rats from threeexperiments for 60-hour exposure). *, p<0.05; **, p<0.01. RA: room aircontrol.

FIG. 16. T3 decreases hyperoxia-induced myeloperoxidase (MPO) activityin lung tissue. Data are presented as mean±SD. of independentexperiments (four rats from two experiments for 48-hour exposure; fourrats from three experiments for 60-hour exposure). *, p<0.05; **,p<0.01. RA: room air control.

FIG. 17. T3 decreases lung neutrophils (MPO-positive cells) afterhyperoxia. Lung tissues were obtained at 60 hours after hyperoxiaexposure with/without T3 injections. Immunostaining was performed withprimary: anti-MPO antibody. Black dots represent MPO-positive cells.

FIG. 18. T3 injection reduces morphologic hyperoxic lung injury. Ratlung tissues at 60 hours of hyperoxia exposure with/without T3injections were stained with hematoxylin, and examined by lightmicroscopy. (A) Control: room air; (B) Hyperoxia; (C) Hyperoxia+T3.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

This disclosure describes compositions that include triiodothyronine(T3) is effective for treating pulmonary edema and/or lung inflammationsuch as, for example, processes that occur in acute respiratory distresssyndrome (ARDS). The T3 compositions are formulated to be administereddirectly into the lung, whether in a liquid form or as an aerosol. Thisdisclosure further describes methods of treating lung inflammation byadministering a formulation of T3 directly to the nasosinus,intratracheal, intrabronchial, or alveolar space.

The lung is a target tissue of thyroid hormone (TH). Thyroid hormoneaffects lung development, lung function, and repair of injury to lungtissues. In rodents, hypothyroidism impairs clearance of airspace fluidduring lung development, while systemic T3 augments alveolar fluidclearance in adult rat lungs. T3 stimulation of alveolar fluid clearanceoccurs locally and rapidly in the lung. Further, mouse models withknockout of either the thyroid hormone receptor (TR) alpha or beta geneshave altered lung development, and response to stress and injury. Insheep models, preterm lambs showed significant improvement in perinatallung function when thyroxine was added to betamethasone injections.

Clinically, thyroid disorders are associated with diverse pulmonarysymptoms. Both hypothyroidism and hyperthyroidism may cause respiratorymuscle weakness and/or decreased pulmonary function. Hypothyroidismreduces respiratory drive and also can cause obstructive sleep apnea orpleural effusions. Conversely, hyperthyroidism increases respiratorydrive and can cause dyspnea on exertion. Either hypothyroidism orhyperthyroidism, can be associated with idiopathic primary pulmonaryarterial hypertension (IPPAH). Further, treating the underlying thyroiddisorder may reverse pulmonary hypertension, although the exactmechanism involved in the pathogenesis is not established.

At the cellular level, thyroid hormone status affects alveolar number,the number and size of alveolar type II pneumocyte cells, and theirsurfactant production. T3 increases alveolar fluid clearance (AFC) inalveolar epithelial cells through augmented Na,K-ATPase activity. Activesodium resorption is involved in clearing pulmonary (alveolar) edema inlungs at birth, in acute lung injury (ALI), in acute respiratorydistress syndrome (ARDS), and in cardiogenic edema, such as congestiveheart failure. Conversely, reducing T3 levels in the lung can exacerbatealveolar edema.

This disclosure describes methods that involve administering T3 directlyto the nasosinus, intratracheal, intrabronchial, or alveolar airspaceby, for example, spray, inhalation, nebulization, or instillation. Whiledescribed herein in the context of an exemplary embodiment in whichalveolar edema and/or lung inflammation are associated with acuterespiratory distress syndrome (ARDS), the compositions and methodsdescribed herein can be used to treat alveolar edema and/or inflammationof lung tissue regardless of the underlying cause of the inflammation.Exemplary other causes of lung inflammation or alveolar edema that aretreatable using the compositions and methods described herein include,for example, premature birth, chest trauma, congestive heart failure,pre- and/or post-lung transplant, pre- and/or post-lung cancerradiotherapy or chemotherapy, pneumonia, sepsis, smoking (whethertobacco or THC), exposure to pollutants (whether environmental oroccupational, e.g., asbestosis, silicosis, berylliosis, Coal Worker's,pneumoconiosis, gas exposure, thermal injury, or other pneumoconiosis),hypersensitivity pneumonitis, reactive or obstructive lung diseases(e.g., asthma, chronic bronchitis, reactive airway dysfunction syndrome,or other reactive airway diseases), aspiration chemical pneumonitis orpneumonia, pneumonia or an infection of nasosinus, intratracheal,intrabronchial or alveolar airspace (e.g., bacterial, viral, fungal),connective tissue diseases (e.g., rheumatoid arthritis, systemic lupuserythematosus, scleroderma, sarcoidosis, and other related diseases),Wegener's granulomatosis, Good pasture disease, acute or chroniceosinophilic pneumonia, medication-related lung injury (e.g., injuryfrom use of amiodarone, bleomycin, busulfan, mitomycin C, methotrexate,apomorphine, nitrofurantoin, or other pneumotoxic drugs), cryptogenicorganizing pneumonia, Churg-Strauss syndrome, or congenital orstructural lung disease (e.g., cystic fibrosis, bronchiectasis.

Acute respiratory distress syndrome (ARDS) is characterized byhemorrhagic inflammatory pulmonary edema with decreased alveolar fluidclearance (AFC) and high mortality. Triiodothyronine (T3) acts onalveolar type II pneumocytes to augment their Na,K-ATPase activity,thereby promoting edema fluid clearance and augmenting oxygen diffusioninto the capillaries. T3 is inactivated by enzyme iodothyroninedeiodinase type-III (D3). Most patients with ARDS have reduced abilityto clear alveolar edema fluid. Moreover, a slower rate of alveolar fluidclearance is associated with higher mortality and longer requirement forsupport with mechanical ventilation. Thus, improving alveolar fluidclearance can improve outcomes for patients with ARDS. This disclosurereports that D3 expression and activity are elevated in early ARDS humanlung tissue. D3 induction in early ARDS is accompanied by local lung T3inactivation, resulting in a decrease in lung T3 concentration in lungtissue. Given that T3 stimulates alveolar fluid clearance, D3-inducedinactivation of lung T3 may impede alveolar fluid clearance in ARDS,contributing to the degree of alveolar flooding with fluid and thepersistent hypoxemia.

ARDS lung tissue samples showed characteristic diffuse alveolar damagewith proteinaceous alveolar filling within the air spaces, hyalinemembrane formation and inflammatory cells in the interstitium (FIGS. 1A,1D, 1G). Control tissue samples demonstrated normal histologicarchitecture of the alveoli and interstitium (FIGS. 1B, 1E, 1H).Immunohistochemical localization of D3 in ARDS tissue revealed highlevel D3 expression in alveolar type II pneumocytes (FIGS. 1A, 1D),spindle-shaped interstitial cells, and capillary endothelial cells (FIG.1G). Normal control tissue demonstrated much less D3 antibody stainingin all of these cell types (FIGS. 1B, 1E, 1H). As a specificity control,removal of primary antibody resulted in no significant D3 staining ofany tissue sections (FIGS. 1C, 1F, 1I).

To determine whether the increase in D3 expression in ARDS lungs wasassociated with increased enzymatic activity, D3 enzyme activities weremeasured in early ARDS (n=3), late ARDS (n=5), and control (n=4) lungsamples. Lung D3 enzyme activity was approximately 11.3 times higher inearly ARDS versus normal control tissue (1.57 vs. 0.14±SEM fmol/mg/min,p<0.0001) (FIG. 2A). D3 activity was approximately 2.5 times higher inlate ARDS vs control lungs (0.34 vs 0.14 fmol/mg/min, p=0.29, n.s.).Lung T3 levels were 65% and 77% lower in early and late ARDS,respectively, compared to control lung levels (FIG. 2B). Together, thesedata demonstrate that D3 expression and activity are markedly induced inthe lungs of early ARDS patients and the increased D3 is associated withlocal reduction in total tissue T3. These data connect the role of T3 inpromoting alveolar fluid clearance (AFC) in ARDS and the role of D3causing T3 inactivation in hypoxic, inflammatory conditions.

In lung injury, the permeability of the alveolar epithelium and thecapillary endothelium are increased, allowing ready transcapillarydiffusion of proteins, solutes, and fluid into the interstitium andalveolar space. Resorption of interstitial edema and, particularly,alveolar edema fluid is crucial for efficient gas exchange in thealveoli. Alveolar fluid clearance is driven by active alveolarepithelial sodium resorption across the alveolar epithelial barrierthrough combined action of basolateral Na,K-ATPase pump and apicalsodium transport proteins.

In both normal and in injured rat lungs, T3 instillation significantlyincreases alveolar fluid clearance. Local and/or systemic inflammationmay initiate D3 induction in the ARDS lung. Acute bacterial infectionsand/or infarction/ischemia can trigger D3 expression. The ARDS in thepatients of this study resulted from a variety of etiologies, includingpneumonia (viral or bacterial), sepsis, trauma, and post-surgical lunginjury, all with inflammation as the likely common pathway to D3induction and subsequent T3 depletion. Decreased local T3 concentrationin the ARDS lung impedes alveolar fluid clearance. The decreasedalveolar fluid clearance impairs oxygen diffusion and exacerbateshypoxemia, a hallmark of ARDS. At baseline in normal circumstances, fivepercent of total-body oxygen uptake is consumed for the mechanics ofrespiration and lung function. In critical illness, such as respiratoryfailure, the metabolic requirements of the lung usually aresignificantly increased to maintain adequate oxygenation andventilation. In ARDS, systemic and local inflammation likely augmentsystemic and local expression of D3, lowering T3 level anddownregulating lung metabolism at a time when accelerated function maybe desired. Because all other organs depend on the lung gas exchange foroxygen, and because T3 is involved in maintaining alveolar fluidclearance and diffusing capacity, T3 deficiency in the lung has adeleterious effect.

T3 instillation augments alveolar fluid clearance in normal andhyperoxia-injured lung tissue. Hyperoxia-induced lung injury (HALI) is awell-established animal model of acute lung injury. Hyperoxia-generatedreactive oxygen species (ROS) lead to alveolar epithelial andendothelial cell death by apoptosis and necrosis, contributing to lunginjury. The molecular basis of oxygen toxicity is mediated by freeradical ROS (reactive oxygen species) derived directly from molecularoxygen and/or derived indirectly from interactions of molecular oxygenwith other species. Oxidants therefore mediate the development of acuteand chronic lung injuries. Thyroid hormone affects antioxidant defensesof both adult and developing rat brain and lung. This disclosurepresents data evaluating the effects of systemic T3 supplementation onlung inflammation and injury in the HALI model.

Hyperoxia decreased serum total T3 levels. Critical illness often causesthe euthyroid sick syndrome or nonthyroidal illness, with decreases ofserum total and free T3 concentrations. FIG. 13 shows data measuring thetotal serum T3 levels in rats exposed to 95% oxygen for 60 hours with orwithout intraperitoneal T3 supplementation (50 μg/kg bodyweight/24hours). Hyperoxia significantly reduced the serum total T3 compared withnormoxic room air (RA) rats (RA: 82.97±14.4399, hyperoxia: 53.9±11.2953,p=0.00043), and supplementation augmented serum total T3.

T3 decreased the hyperoxia-induced increases in lung edema andbronchoalveolar lavage fluid (BALF) protein concentration. Adult ratsexposed to 95% oxygen for 60 hours have substantial lung injury asdocumented by increases in BALF protein concentration, permeability, andlung edema. Hyperoxia induces increased wet-to-dry lung weight ratioscompared to normoxic rat lungs (6.49±0.27 vs. 5.3±0.16, respectively,p=0.004). FIG. 14A shows that 60 hours of hyperoxia again markedlyincreased the wet-to-dry lung weight ratio compared with room air (O₂6.61±0.60 vs. RA 4.82±0.14). Treatment with intraperitoneal T3 (12.5μg/kg body weight injected each 12 hours) significantly decreased thishyperoxia-induced increase (O₂: 6.61±0.604; O₂ with T3: 5.82±0.197,p=0.0495 vs O₂ treatment) (FIG. 14A). Hyperoxia also increased markedlythe BALF protein concentration at both the 48 hour and 60 hour timepoints, and T3 administration attenuated significantly the hyperoxicincreases in the BALF protein concentration at both time points (FIG.14B). Thus, T3 supplementation decreased the extent of pulmonary edemaand the dysfunction of the alveolar epithelial barrier caused byhyperoxia.

T3 reduced the hyperoxic increases of BALF cellularity and lung tissueneutrophil accumulation. In adult rat lungs 95% oxygen exposureaugmented the number of inflammatory cells in BALF. Indeed, 48 hours or60 hours exposure to 95% oxygen markedly increased the number ofbronchoalveolar lavage (BAL) cells compared to the control rats in theroom air. Most of the BAL cells were mononuclear cells and macrophages,but differential cell counts were not performed. T3 administrationduring hyperoxia significantly reduced the BALF cell numbers at bothtime points compared with their hyperoxia alone counterparts (FIG. 15).

Neutrophil infiltration into the lung is a component of lunginflammation that often is a prelude to and component of lung injury.However, relatively few of the BALF cells after hyperoxia wereneutrophils (data not shown). The effects of T3 on lung tissueneutrophils under hyperoxia were directly assessed in two ways:measurement of myeloperoxidase (MPO) activity in lung homogenates andimmunostaining of the lungs for MPO. Although lung MPO activity was notaltered by hyperoxia at 48 hours (FIG. 16, left panel), lung MPOactivity was markedly increased at 60 hours of hyperoxia compared toroom air controls (FIG. 16, right panel). T3 supplementationsignificantly reduced the hyperoxia-induced MPO activity (FIG. 16, rightpanel). Similarly, cytochemistry demonstrated that the T3-injected ratsexposed to 95% oxygen for 60 hours displayed fewer MPO-positive cells inthe lungs compared with hyperoxic lungs without T3 supplementation,confirming the MPO activity results (FIG. 17). Systemic T3supplementation inhibited the hyperoxia-induced neutrophil accumulationwithin the lung tissue.

T3 reduced the hyperoxia-induced morphologic lung injury.Histopathological evaluation of lung sections also was performed toassess qualitatively whether T3 reduced hyperoxic lung injury. Asexpected, hyperoxia alone caused alveolar septal thickening, lung edema,and alveolar inflammatory cells (FIGS. 18A and 18B). In contrast,systemic T3 supplementation led to the persistence of virtually normallung morphology (FIG. 18C). The striking difference in lung histologyfurther demonstrated that T3 supplementation significantly attenuatedhyperoxic lung injury.

Thus, this disclosure provides data showing that T3 administrationconcomitant with the hyperoxic exposure significantly decreased theseverity of hyperoxia-induced rat lung injury, with reduced neutrophilaccumulation in the lungs, diminished lung edema, and less breakdown ofthe alveolar epithelial permeability barrier. T3 supplementationsignificantly reduced, but did not completely eliminate the hyperoxiclung injury and inflammation. These results strongly suggest aprotective anti-inflammatory effect of T3 against hyperoxic lung injury.

Hyperoxia exposure decreased the serum total T3 concentration (FIG. 13).Intraperitoneal injections of T3 restored the serum total T3 (FIG. 13)and significantly decreased the hyperoxia-mediated lung injury in adultrats, as measured by a reduction in the increases of: (1) the ratio ofwet/dry lung weight at 60 hours (FIG. 14A); (2) the BAL:fluid totalprotein concentration at 48 hours or 60 hours after exposure (FIG. 14B);(3) the number of nucleated cells in BAL fluid at 48 hours or 60 hours(FIG. 15); (4) neutrophil accumulation in lung tissue at 60 hours (FIG.16 and FIGS. 17); and (5) the histopathologic diffuse alveolar wallinjury and infiltration with inflammatory cells in rat lungs at 60 hours(FIG. 18). These results demonstrate that T3 reduces hyperoxia-mediatedlung inflammation and injury.

Thyroid hormone has not generally been appreciated as an importantregulator of lung function. However, thyroid hormones have amultiplicity of effects on the lung. For example, thyroid hormoneincreases the number of alveolar type II cells, the number of lamellarbodies, and surfactant release. Systemic or local T3 administrationenhances alveolar edema fluid clearance in both normal andhyperoxia-injured rat lungs and hyperoxia-injured rat lungs. Airspace T3administration rapidly restored the AFC decreased by hyperoxia-inducedlung injury in rat lungs. T3 also significantly increases type IIalveolar epithelial cell Na,K-ATPase activity, consistent with its rolein removing edema fluid from the alveolar space. This action of T3involves coordinated action of both the PI-3 kinase (PI3K)/Akt andERK1/2 pathways. The PI-3K/Akt pathway is involved in many cellresponses to stress, including inflammation.

Instilled T3 also enhances survival of alveolar Type II cells exposed tohyperoxic stress. Hyperoxic exposure causes both apoptosis and necrosisof alveolar epithelial cells. Survival of alveolar type II (AT2) cellsis important for recovery after oxidant-induced lung damage. Thyroidhormone (T3) reduced hyperoxia-induced lung inflammation in adult ratsexposed to >95% oxygen in vivo.

Hyperoxia-induced acute lung injury (HALI) in animals is awell-established model of acute lung injury and ARDS. Prolonged exposureto hyperoxia leads to the generation of excessive reactive oxygenspecies, causing injury and death of alveolar endothelial and epithelialcells accompanied by high alveolar levels of pro-inflammatory cytokinesand excessive leukocyte infiltration.

Alveolar epithelial and endothelial cells maintain the integrity of thealveolar-capillary barrier and defend against oxidative injury.Prolonged exposure to hyperoxia generates excessive reactive oxygenspecies (ROS), damaging cells by overwhelming redox homeostasis. Thenuclear factor erythroid 2-related factor 2 (Nrf2) transcription factorprotects cells against oxidative insults and chemical carcinogens bycoordinated transcriptional activation of a panel ofantioxidant/detoxifying enzymes, including heme oxygenase-1 (HO-1),glutathione-S-transferase (GST), NAD(P)H:quinone oxidoreductase-1(NQO-1), glutamate cysteine ligase, peroxiredoxin 3, peroxiredoxin 6,manganese superoxide dismutase, and catalase. Genetic ablation of Nrf2enhances lung injury induced by hyperoxia, while amplification ofendogenous Nrf2 activity attenuates HALI. Increased expression ofantioxidant enzymes and phase 2 detoxifying enzymes in lung epithelialcells protects against the damage caused by hyperoxia-generated ROS.Nrf2-regulated HO-1 confers cytoprotection against cell death in variousmodels of lung injury by inhibiting apoptosis. Nrf2 activation promotesalveolar cell survival during oxidative stress.

T3 increased the number of viable AT2 cells after 72 hours of exposureto 90% oxygen. In vivo hyperoxia causes rat lung inflammation and injurysimilar to early phase ARDS and in vitro hyperoxic exposure is a widelyused model to study alveolar epithelial cell injury and function inARDS. Using MDCK cells, cell density determined the balance ofapoptosis, necrosis, and cell proliferation during hyperoxia exposure.In vivo hyperoxia exposure dramatically decreases serum T3 while T3supplementation attenuates hyperoxia-induced lung inflammation. FIG. 7shows that T3 protects alveolar epithelial cells from hyperoxic damageand increases their survival. Rat adult AT II-like cell line RLE-6TNwere exposed to 90% oxygen for 72 hours in the presence or absence of T3or RT3 (inactive thyroid hormone) and the number of viable cells wasmeasured by trypan blue exclusion. Hyperoxia dramatically decreased thenumber of surviving AT2 cells by almost 75% compared to room air (21%oxygen) culture conditions (FIG. 7A). T3 significantly increased thenumber of surviving cells under hyperoxia stress by ˜2.5-fold comparedwith hyperoxia control. In contrast, rT3 had no role in cell survival.The protective effect of T3 was observed across a range of pharmacologicconcentrations (10-7 to 10-5 M) (FIG. 7B). When the AT2 cells that hadbeen exposed to hyperoxia for 72 hours with or without exogenous T3 wereallowed to recover in room air for an additional 72 hours (with nosupplemental T3), the protective effect of T3 on viable AT2 cell numberpersisted and was of similar magnitude (FIG. 7C). The beneficial effectof having T3 at pharmacologic concentration present during hyperoxia wasmanifested as more surviving AT2 cells even after a recovery period inroom air. These results demonstrated that T3 significantly protected AT2cell survival during hyperoxia.

T3 augmented recovering AT2 cell number after hyperoxic injury. Alveolarepithelial recovery after lung inflammation and injury promotes therecovery of patients with ARDS. FIG. 8 shows that T3 positively impactedAT2 cell recovery after hyperoxia-induced damage. RLE-6TN cells wereexposed to 90% oxygen for 24 hours or 48 hours. Then the cells recoveredin 21% O₂/5% CO₂ in the presence or absence of T3 or rT3 for 48 hours.After either 24 or 48 hours of injury (FIG. 8A and FIG. 8B,respectively) followed by recovery, T3 significantly increased thenumber of viable AT2 cells at 48 hours of recovery, whereas rT3 had noprotective effect. Thus T3 also has a beneficial effect on AT2 cellrecovery from hyperoxia if it is present only during the recovery phase.

T3 increased Nrf-2 protein expression and nuclear translocation underhyperoxia stress. The transcription factor Nrf2 (NF-E2-related factor 2)promotes cellular homeostasis, especially during exposure to chemical oroxidative stress. Nrf2 regulates the basal and inducible expression of amultitude of antioxidant proteins, detoxification enzymes, andxenobiotic transporters. FIG. 9 shows that T3 increases Nrf2 activity.Total cellular and nuclear Nrf2 protein were assessed at 24 hours ofhyperoxia exposure. Hyperoxia decreased the total Nrf2 proteinexpression, surprisingly without changing the nuclear Nrf2 proteinlevel. T3 treatment during hyperoxia significantly increased both totalcellular and nuclear Nrf-2 protein expression (FIG. 9).

T3-induced increase in HO-1 is required for T3-increased RLE-6TN cellsurvival in hyperoxia. Heme oxygenase-1 (HO-1) is an anti-inflammatory,antioxidative, and cytoprotective enzyme that is regulated by theactivation of the major transcription factor Nrf2. HO-1 is the inducibleisoform of the first and rate-limiting enzyme of heme degradation andits induction protects against oxidative stress and apoptotic celldeath. Desoxyrhapontigenin upregulates Nrf2-mediated heme oxygenase-1expression in macrophages and inflammatory lung injury. FIG. 10 showsthat T3 altered HO-1 expression in AT2 cells during hyperoxia. RLE-6TNcells were exposed to 90% O₂ for 24 hours and then cell survival andHO-1 expression were measured. The impact of the HO-1 inhibitor, tinprotoporphyrin, on cell survival also was determined. T3 treatmentsignificantly enhanced total cellular protein of HO-1 during hyperoxia(FIG. 10A). Tin protoporphyrin had no effect on AT2 cell number in roomair (FIG. 10B), but it blocked the T3-caused increase in cell survivaland augmented cell death (FIG. 10C). Thus, T3 augments HO-1 expressionduring hyperoxia and HO-1 upregulation facilitates the protective effectof T3 on AT2 cell survival.

PI3-kinase activity medaites the T3 effects on AT2 cell survival, Nrf2activity, and HO-1 expression. The PI3K/Akt is an anti-apoptoticsurvival pathway and is regulated by a number of receptor-dependentmechanisms. T3 stimulates PI3K activity and activation of this pathwaypromotes T3-induced increases of Na,K-ATPase activity and plasmamembrane expression. In vascular endothelium, PI3K activation increasesHO-1 expression, while PI3K activation augments Nrf2 protein levels andHO-1 activation in other cell types. To detect whether the PI3K/Aktpathway is required for the T3 protective effects on alveolar cellsurvival, Nrf2 activity and HO-1 protein levels during hyperoxia,RLE-6TN cells were cultured for 72 hours in hyperoxia in the presence of10⁻⁶M T3 and/or 100 nM wortmannin. Wortmannin blocked the T3-inducedcell survival during hyperoxia, and resulted in death of almost all thecells (FIG. 11).

FIG. 12 shows the effect of PI3K on activation of Akt byphosphorylation. The RLE-6TN cells were exposed to hyperoxia for 24hours and T3 was added for 20 minutes to some cells. Hyperoxia alone didnot alter the quantity of total or phospho-Akt compared to room aircontrol cells, whereas T3 augmented phosphorylation of Akt at Ser473(FIG. 12A) compared with room air control and hyperoxia alone. PI3kinase is activated by T3 during hyperoxia and activation of thispathway facilitates the protective effect of T3 on AT2 cell survival. Inaddition, the PI3K inhibitor wortmannin also blocked the T3-inducedincreases in both Nrf2 total cellular protein and nuclear protein (FIG.12B), and total cellular amount of HO-1 protein (FIG. 12C). Thus, thePI3-kinase pathway is activated by T3 during hyperoxia and activation ofthis pathway is involved in the beneficial effects of T3 on thecytoprotective effectors HO-1 and Nrf2 and on AT2 cell survival.

Recovery of the alveolar epithelial barrier is a component of therecovery from ARDS. Protecting alveolar epithelial cells from dyingduring injury and/or augmenting re-epithelialization are strategies tospeed healing. Thyroid hormone has not been conceived as a determinantof lung healing, but serum T3 levels are decreased in both humans andrats with acute lung injury. Lung tissue T3 levels also are markedlydecreased. AT2 cells respond to pharmacologic and physiologicconcentrations of T3 through both PI3K/Akt and MAP kinase mediatedpathways that increase Na,K-ATPase activity and also increases alveolarfluid clearance in vivo.

This disclosure provides data showing that T3 at pharmacologicconcentrations increases AT2 cell survival during hyperoxia andaccelerated the recovery in AT2 cell number after hyperoxia. Theseeffects were associated with activation of PI3 kinase and Nrf2 and withupregulation of HO-1 expression. The cytoprotective effects of T3 wereabrogated when PI3K activation was blocked by wortmannin or when HO-1expression was blocked by tin protoporphyrin. These findings suggestthat T3 augmentation in the lung augments alveolar epithelial repair.

In many studies, exposure of lung epithelial cells to hyperoxia for60-72 hours causes cell death. However, this disclosure provides datashowing that treatment of RLE-6TN cells with T3 prior to exposure tohyperoxia increased viable cell numbers compared to untreated controls.The effect of T3 on cell number may be due, at least in part, topreserving cell proliferation, diminishing cell death by necrosis,diminishing cell death by apoptosis, and/or other mechanisms. T3augments proliferation in some selected cell types via PI3K pathwayactivity (human glioma, human pancreatic insuloma), but inhibitsproliferation in others (human proximal tubule cell line (HK2) and renalcancer cell lines (Caki-2, Caki-1)). Past studies of cultured AT2 cellshave not demonstrated stimulation of proliferation by thyroid hormones.The effect of T3 on cell proliferation appears, therefore, to be celltype specific. The effect of thyroid hormones on cell death across avariety of cell types has not been carefully examined.

Hyperoxic exposure of AT2 cells causes a block in cell proliferation andaugments cell death by a combination of necrosis and apoptosis. Thisdisclosure presents data that shows the number of surviving AT2 cellsunder hyperoxic stress in the presence of T3 was 2.5-fold higher than inhyperoxia alone (FIG. 7), most likely due to a strong cytoprotectiverole of T3 in hyperoxia. Regeneration and/or restoration of alveolarcells after lung injury improves restoration of normal lung structureand function. T3 also enhanced RLE-6TN cell recovery from hyperoxicinjury (FIG. 8).

Nrf-2 is involved in regulating antioxidant defense. Nrf-2 activation inresponse to oxidant exposure is involved in inducing severalantioxidative and cytoprotective enzymes that mitigate cellular stress.In animal studies, Nrf-2 expression is involved in protection againsthyperoxic lung injury. Hydrogen gas, a scavenger of reactive oxygenspecies (ROS), reduces hyperoxic lung injury via Nrf-2 pathway. Lungepithelial and endothelial cell death is main characteristics ofhyperoxic lung injury. This disclosure presents data showing that T3 inhyperoxia increases Nrf2 expression and nuclear accumulation (FIG. 9),indicating that T3 increases Nrf2 activity in alveolar epithelial cells.Nrf2 cytosol-to-nuclear translocation may represent a novelcytoprotective mechanism of T3 to limit free radical or electrophiletoxicity.

Nrf2, as a sensor for oxidative/electrophilic stress, is constantlydegraded through a Keap 1/Cullin3/Ring Box 1 (Cul3/Rbx1) E3 ubiquitinligase complex pathway in the cytoplasm. When a cell is exposed tooxidative stress, Nrf2 dissociates from the Keap 1 complex, stabilizesand translocates into the nucleus, leading to activation of ARE-mediatedgene expression. Several protein kinases including PKC, MAPK/ERK, p38,PERK, and PI3K/Akt can activate Nrf2, while GSK-3beta, whose activitycan be inhibited by Akt-mediated phosphorylation at Ser9, inactivatesNrf2. GSK-3beta phosphorylates Fyn, a tyrosine kinase, leading tonuclear localization of Fyn. Fyn phosphorylates tyrosine 568 of Nrf2,resulting in nuclear export of Nrf2, binding with Keap 1, anddegradation of Nrf2. 60-minute exposure to hyperoxia increases Nrf2activity via PI3K/Akt in lung epithelial cells. This disclosure providesdata showing that the PI3K inhibitor wortmannin abolished T3-inducedincrease in nuclear levels of Nrf2 in hyperoxia (FIG. 12B), suggestingthat PI3K activation by T3 is involved in Nrf2 nuclear translocation.

Heme oxygenase-1 (HO-1) is an antioxidant enzyme that mediatescytoprotection against oxidative stress. HO-1 prevents hyperoxia-inducedlung endothelial death in a mouse model. Up-regulation of Nrf2-mediatedheme oxygenase-1 expression by eckol in Chinese hamster lung fibroblast(V79-4) cells via PI3K/Akt pathway. This disclosure presents datademonstrating that T3 increased HO-1 expression in alveolar epithelialcells under in hyperoxic conditions (FIG. 10A). HO-1 inhibitor, TIN,blocked T3-induced increase in cell survival (FIG. 10C). These resultsindicated that HO-1 facilitates the cytoprotective effect of T3 inhyperoxia.

In summary, upregulation of antioxidative and cytoprotective geneexpression by Nrf-2 mediates alveolar epithelial cell survival and/ordecreases cell death in response to hyperoxia stress. T3 increasesalveolar epithelial cell survival and speeded up cell recovery fromhyperoxic injury. These cytoprotective effects of T3 involve activationof Nrf2 and upregulation of HO-1 gene expression via T3-activated PI3K.

This disclosure therefore describes compositions and methods formaintaining or restoring T3 levels in a subject. Typically, the subjectis a human. The compositions generally include T3 in a modifiedformulation. The modified formula results in a well-tolerated instillantwith limited systemic exposure. Instilled T3, in the modifiedformulation, can be effective for reducing—in some cases,eliminating—lung inflammation (e.g., associated with lung transplant,radiotherapy or chemotherapy), augmenting pulmonary edema fluidclearance, diminishing lung injury, and/or treating lung inflammationassociated with pulmonary disease or injury (e.g., ARDS). T3,administered directly into the lungs rather than systemically, is a safeprophylactic and/or therapeutic treatment for lung inflammation.

The conventional T3 liquid formulation is FDA-approved for clinical useto treat myxedema coma/precoma by intravenous administration directlyinto the bloodstream. The conventional formulation is not pH adjustedbecause intravenous administration to the bloodstream causes the T3 tobe rapidly diluted and buffered by the buffering capacity of serumproteins and other blood components. When the conventional T3formulation is administered directly into the lung, its concentration isnot diluted immediately and the degree of buffering capacity of thelung, both tracheobronchial tree and alveolar-lining fluids isuncertain. Thus, instilling T3 into the airway and lung directly fromthe manufacturer's vial—i.e., without adjusting the pH—is extremelytoxic to rat lung mucosa and airspaces.

The modified formulation described herein includes T3, adjusted toneutral pH (5.5-8.5). The T3 may be formulated with any suitablepharmaceutically acceptable carrier. As used herein, “carrier” includesany solvent, dispersion medium, vehicle, coating, diluent,antibacterial, and/or antifungal agent, isotonic agent, absorptiondelaying agent, buffer, carrier solution, suspension, colloid, and thelike. The use of such media and/or agents for pharmaceutical activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient or is known tobe injurious to lung tissue, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients also can be incorporatedinto the compositions. As used herein, “pharmaceutically acceptable”refers to a material that is not biologically or otherwise undesirable,i.e., the material may be administered to an individual along with T3without causing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained.

T3 may therefore be formulated into a pharmaceutical composition. Thepharmaceutical composition may be formulated in a variety of formsadapted for delivery to the nasosinus, intratracheal, intrabronchial, oralveolar space. A pharmaceutical composition can be administered to amucosal surface, such as by administration to, for example, respiratorymucosa (e.g., by spray, aerosol, nebulization, or instillation). Acomposition also can be administered via a sustained or delayed release.Sustained or delayed released may be accomplished through conventional,general technologies for sustained or delayed drug delivery. Sustainedrelease also may be accomplished by combining the T3 with a second drugthat inhibits a mechanism that degrades or clears T3 from the lung. Oneexemplary second drug is an inhibitor of D3, such as, for example,iopanoic acid.

The modified formulation described herein includes T3, adjusted toneutral pH. As used herein, the term “neutral pH” refers to a pH that ispH 7.0+1.5—i.e., a pH of 5.5 to 8.5. In some embodiments, theformulation may be buffered to a minimum pH of at least 5.5, at least6.0, at least 6.5, at least 7.0, or at least 7.5. In some embodiments,the formulation may be buffered to a maximum pH of no greater then 8.5,no greater then 8.0, no greater then 7.5, no greater than 7.0, or nogreater than 6.5. In some embodiments, the formulation may be bufferedto a pH that falls within a range having endpoints defined by anyminimum pH listed above and any maximum pH listed above that is greaterthan the minimum pH. Thus, for example, the formulation may be bufferedto a pH of from 5.5-8.5, such as, for example, a pH of 5.5-7.0, a pH of6.0-8.0, a pH of 6.0-7.0, or a pH of 6.5-7.5.

Thus, T3 may be provided in any suitable form including, but not limitedto, a solution, a suspension, an emulsion, a spray, an aerosol, or anyform of mixture. The composition may be delivered in formulation withany pharmaceutically acceptable excipient, carrier, or vehicle.Exemplary suitable excipients include, but are not limited to, dextroseand ammonium hydroxide. For example, the formulation may be delivered ina dosage form suitable for direct delivery to the lungs such as, forexample, an aerosol formulation, a non-aerosol spray, a solution, aliquid suspension, and the like. The formulation may further include oneor more additives including such as, for example, an adjuvant, acolorant, a fragrance, a flavoring, and the like.

A formulation may be conveniently presented in unit dosage form and maybe prepared by methods well known in the art of pharmacy. Methods ofpreparing a composition with a pharmaceutically acceptable carrierinclude the step of bringing T3 into association with a carrier thatconstitutes one or more accessory ingredients. In general, a formulationmay be prepared by uniformly and/or intimately bringing the activecompound into association with a liquid carrier, a finely divided solidcarrier, or both.

The amount of T3 administered can vary depending on various factorsincluding, but not limited to, the weight, physical condition, and/orage of the subject; the particular clinical signs or symptoms exhibitedby the subject; the type or cause of lung inflammation or pulmonaryedema; and/or the method of administration. Thus, the absolute amount ofT3 included in a given unit dosage form can vary widely, and dependsupon factors such as the species, age, weight and physical condition ofthe subject, and/or the method of administration. Accordingly, it is notpractical to set forth generally the amount that constitutes an amountof T3 effective for all possible applications. The physiologicallyactive T3 concentration at the cellular level has been determined andvaries depending upon the cell type and the specific hormonal targeteffect. Dosing of T3 can be designed to achieve either physiologic orpharmacologic local tissue levels. Those of ordinary skill in the art,however, can determine the appropriate amount with due consideration ofsuch factors.

For example, T3 may be administered to treat pulmonary edema or lunginflammation at the same dose and frequency for which T3 has alreadyreceived regulatory approval. In other cases, T3 may be administered fortreating alveolar edema or lung inflammation at the same dose andfrequency at which the drug is being evaluated in clinical orpreclinical studies. One can alter the dosages and/or frequency asneeded to achieve a desired level of T3. Thus, one can usestandard/known dosing regimens and/or customize dosing as needed.However, the primary active form of T3—i.e., the form in which it hasthe greatest physiological activity—is when the T3 is “free”—e.g., notbound to large proteins such as albumin. Therefore, the physiologiceffect of a given amount of T3 also may be influenced by the proteinsand other aspects of the environment that it is introduced into. Thus, asmaller amount of T3 may be required to achieve an effective drugdelivered dose for the methods described herein—i.e., in the “free”state and delivered directly to the nasosinus, intratracheal,intrabronchial, or alveolar airspace—than the dose of T3 receivingregulatory approval from treating other conditions by intravenousdelivery.

In some embodiments, the method can include administering sufficient T3to provide a deposited dose of, for example, from about 0.5 μg to about2.0 mg to the subject, although in some embodiments the methods may beperformed by administering T3 in a dose outside this range. In some ofthese embodiments, the method includes administering sufficient T3 toprovide a deposited dose of from about 5 μg to about 50 μg to thesubject. On a μg/kg basis, the calculated administered T3 dose toachieve physiologic effects could range from as low as 2 ng/kg to 1mg/kg. As one example, a 50 μg dose can provide a μg/kg dosage range offrom about 0.03 μg/kg (to a 160 kg person) to as high as 25 μg/kg (to a2 kg preterm infant. In many instances, however, dosing on a μg/kg basisis less relevant since direct instillation to lung tissue is not assubject to systemic dilution as, for example, intravenousadministration. Lung size in adults does not vary significantly withweight, so mass of T3 delivered is often the more relevant measure of anappropriate dose.

As used herein, the term “deposited dose” or “lung-delivered” doserefers to the amount of T3 deposited to the surface of the respiratorytract. For instillation, the deposited dose is essentially the full dosebeing instilled. In an aerosol or nebulized formulation, however, thedeposited dose is conventionally 10% or less of the drug beingaerosolized or nebulized. 90% of the drug is expected to be lost in thedelivery apparatus and/or exhaled. This may be greater in the injuredARDS lung. Thus, one may aerosolize or nebulize 500 μg of T3 to achievean aerosolized or nebulized deposited dose of 50 μg. The use the term“deposited dose” or “lung-delivered” dose normalizes the dose acrossdifferent routes of administration.

A sufficient deposited dose or lung-delivered dose can provide deliveryof a minimum amount of T3 of at least 5 ng such as, for example, atleast 100 ng, at least 1 μg, at least 10 μg, at least 50 μg, at least100 μg, at least 250 μg, at least 500 μg, at least 1 mg, at least 1.5mg, at least 2 mg, at least 5 mg, at least 10 mg, at least 15 mg, atleast 20 mg, or at least 25 mg.

A sufficient deposited dose or lung-delivered dose can provide deliveryof a maximum amount of T3 of no more than 50 mg such as, for example, nomore than 30 mg, no more than 20 mg, no more than 15 mg, no more than 10mg, no more than 5 mg, no more than 4 mg, no more than 3 mg, no morethan 2 mg, no more than 1.5 mg, no more than 1 mg, no more than 500 μg,no more than 300 μg, no more than 200 μg, no more than 100 μg, no morethan 50 μg, no more than 30 μg, no more than 20 μg, or no more than 10μg.

A sufficient deposited dose or lung-delivered dose also can becharacterized by any range that includes, as endpoints, any combinationof a minimum deposited dose or lung-delivered dose identified above andany maximum deposited dose or lung-delivered dose identified above thatis greater than the minimum deposited dose or lung-delivered dose. Forexample, in some embodiments, the deposited dose or lung-delivered dosecan be from 1 μg to 1.5 mg such as, for example, from 5 μg to 50 μg.

In some embodiments, T3 may be administered, for example, from a singledose to multiple doses per day, although in some embodiments the methodcan be performed by administering T3 at a frequency outside this range.When multiple doses are used within a certain period, the amount of eachdose may be the same or different. For example, a dose of 50 μg in a daymay be administered as a single dose of 50 μg, two 25 μg doses, or inmultiple unequal doses. Also, when multiple doses are used within acertain period, the interval between doses may be the same or bedifferent. In certain embodiments, T3 may be administered from aboutonce per day, four times per day, or continuously.

In some embodiments, T3 may be administered, for example, from a singledose to a duration of multiple days, although in some embodiments themethod can be performed by administering T3 for a period outside thisrange. In certain embodiments, T3 may be administered once, over aperiod of three days, or over a period of seven days. In certainembodiments, T3 may be administered from about once per day, four timesper day, or continuously. Usually, thyroid hormone replacement for humanclinical hypothyroidism is given daily with either thyroxine T4 orcombination T4 and T3. A recent study using a single oral dose of 50micrograms of liothyronine resulted in peak serum concentration at 2.5hours with a mean half-life of 22.5 hours. There was a lag between thepeak serum concentration and the physiologic effect of increased heartrate at five hours. (Jonklaas et al., Ther Drug Monit. 37(1): 110-118,2015). For acute severe human illness with myxedema coma, there is awide recommended frequency of intravenous T3 administration, from everyfour hours to every 12-24 hours. Thus, in some embodiments, T3 may beadministered once daily by intratracheal instillation at escalatingdoses with frequent physiologic measurement of hemodynamic parametersand less frequently extravascular lung water (EVLW).

Treating alveolar edema, lung inflammation, or associated conditions canbe prophylactic or, alternatively, can be initiated after the subjectexhibits the onset of pulmonary edema or lung inflammation or theassociated symptoms or clinical signs of a condition. Treatment that isprophylactic—e.g., initiated before a subject experiences an event(e.g., cancer radiotherapy) or manifests a symptom or clinical sign ofthe condition (e.g., while an infection remains subclinical)—is referredto herein as treatment of a subject that is “at risk” of having thecondition. As used herein, the term “at risk” refers to a subject thatmay or may not actually possess the described risk. Thus, for example, asubject “at risk” of infectious condition is a subject present in anarea where other individuals have been identified as having theinfectious condition and/or is likely to be exposed to the infectiousagent even if the subject has not yet manifested any detectableindication of infection by the microbe and regardless of whether thesubject may harbor a subclinical amount of the microbe. As anotherexample, a subject “at risk” of a non-infectious condition is a subjectpossessing one or more risk factors associated with the condition suchas, for example, genetic predisposition, ancestry, age, sex,geographical location, or medical history.

Accordingly, a pharmaceutical composition that includes T3 can beadministered before, during, or after the subject first exhibitspulmonary edema, lung inflammation, or other symptom or clinical sign ofassociated conditions or, in the case of infectious conditions, before,during, or after the subject first comes in contact with the infectiousagent. Treatment initiated before the subject first exhibits pulmonaryedema or lung inflammation or another associated symptom or clinicalsign may result in decreasing the likelihood that the subjectexperiences clinical consequences compared to a subject to whom thecomposition is not administered, decreasing the severity and/orcompletely resolving the lung abnormality. Treatment initiated after thesubject first exhibits clinical manifestations may result in decreasingthe severity and/or complete resolution of pulmonary edema and/or lunginflammation experienced by the subject compared to a subject to whomthe composition is not administered.

For example, hyperoxic injury to rats in vivo and to alveolar type IIcells in vitro is decreased when T3 is given in advance of or coincidentwith injurious hyperoxic exposure. In vitro, alveolar type II cell deathwas significantly reduced. In vivo, lung inflammation, lung injury,neutrophil infiltration and protein leakage into the alveolar space weresignificantly reduced.

Thus, the method includes administering an effective amount of the T3composition to a subject having, or at risk of having, pulmonary edemaor lung inflammation. In this aspect, an “effective amount” is an amounteffective to reduce, limit progression, ameliorate, or resolve, to anyextent, the pulmonary edema or lung inflammation. For example, an“effective amount” of a T3 pharmaceutical composition may increasealveolar fluid clearance, increase the population of alveolar type IIpneumocytes, increase the size of alveolar type II pneumocytes, increaseNa,K-ATPase activity in alveolar epithelial cells, decrease or repairalveolar damage, decrease hypoxemia, and/or decrease in inflammationthroughout the respiratory tract (e.g., nasosinus, intratracheal,intrabronchial and alveolar airspace).

Preliminary studies demonstrate the safety of administering a T3composition via intratracheal instillation. Safety studies are describedin Example 3, below. In a safety study conducted in compliance with GoodLaboratory Practices (GLPs), 0.3 mL (300 μl) was administered to ratsweighing 250-350 grams via intratracheal instillation daily for fiveconsecutive days. No significant complications or changes were observedwith the administration of the materials using a combination ofhistopathologic, physiologic, and laboratory value assessments. All ratsreceived the same dose of T3 (˜2.7 μg in 0.3 mL). The calculated dose ofT3 administered based on Day 1 body weight was 10 μg/kg. The calculateddose based on lung weight was about 1.57 μg/g wet lung weight.

Both male and female rats, prior to and throughout dosing, were observedto have slight/mild porphyrin staining. This likely reflects the mildnon-specific stress induced by 5 days of handling and transportationwith anesthesia and intubation. Since this occurred during theacclimatization and handling period and in the non-T3 treated rats, noneof the observations regarding porphyrin staining can be directlyattributed to treatment with T3.

On average, body weights for all groups tended to decline within thefirst two days of dosing. At the time of terminal euthanasia, bodyweights returned to greater than the pre-dose weights with the exceptionof the T3 females that weighed slightly less than their pre-dose weight.Prior to terminal euthanasia, male toxicity phase animals weighedbetween 267.17 g and 309.60 g with a mean±standard deviation of285.28±11.10 g. Female toxicity phase animals weighed between 240.11 gand 278.44 g with a mean±standard deviation of 258.24±9.79 g.

No clinically significant abnormalities were noted on any animals duringdaily observations from the time of enrollment on study until euthanasiawith the exception of a small number of anesthetic recoverycomplications noted below that varied in severity and cause. There wereno other notable complications throughout the course of the study. Allother surviving animals were in apparent good health prior to terminaleuthanasia. In the toxicity phase of the study there were four salinecontrol animals and one vehicle control animal that died during recoveryfrom anesthesia post-dosing. These deaths occurred approximately 30-60minutes post-dosing and were judged by the veterinary pathologistconducting the post-mortem exam to have been caused by exposure toexcessive temperatures during recovery from anesthesia on heating pads.There were two T3 test animals that died after dose administration. Forone of these deaths, gross pathology findings indicated it to be due tocolon impaction, and in the other animal the cause was undetermined butthought to be due to injury during the instillation procedure. Of allanimals dosed with the test or control articles, only two of seven earlydeaths were animals treated with liothyronine sodium injection. In spiteof these losses, the dosing of spare animals for each group ensured thatthe planned number of animals of each sex/group outlined in the studydesign was achieved.

Hematology and serum chemistry evaluations were performed on alltoxicity phase animals that survived to scheduled termination with theexception of one animal that did not receive hematology analysis due toa clotted sample.

Results of the female hematology showed a mildly increased reticulocytecount and degree of polychromasia in the T3 group compared to thecontrol groups (likely clinically insignificant). There were nostatistical or clinically significant changes in the hematologyparameters in the males. In the chemistry results in the female rats,the only finding was a slight statistical difference in the mean albuminconcentration between the T3 group and control groups (likely clinicallyinsignificant). In males, the only difference in the chemistry valueswas a slight difference in the mean globulin level between the T3 groupand control groups. The differences between the T3 and control groups inthe mean albumin in females and mean globulins are considered clinicallyinsignificant as the changes were minimal and there are no otherclinically significant changes in the laboratory data. The slightlyhigher reticulocyte count in the T3-treated female rat group likely isnot of clinical significance considering the lack of a true referenceinterval and given the absence of a concurrent decrease in hematocrit,hemoglobin, RBC count, abnormal RBC morphology or evidence of anincrease in red blood cell turnover.

In general, the changes noted between the T3 group and control groupsare considered clinically insignificant and not directly related to theT3.

The animal necropsy and gross pathology observations were performed byDr. George Ruth, DVM, PhD, DACVP or Rebecca Rose, DVM, PhD, DACVP. Asidefrom seven early deaths, all toxicity phase animals were euthanized atthe scheduled euthanasia time point. Necropsy was performed on allanimals on the day of death with the exception of the five animals thatdied during recovery from anesthesia, for which necropsy was performedon the day following death. There were no apparent complications duringany of the necropsy procedures, and no gross lesions were detected inthe lungs of any of the animals in this study. Lesions were detected ina total of four animals from among the three treatment groups on theirassigned termination date that probably were associated with terminalintraabdominal injections. No evidence of toxicity was observed grossly,and no clinically significant abnormalities were noted during necropsythat were related to the administration of test or control articles.

Histological evaluation of animals resulted in the following deductions.There were no differences in the organ weight of the lungs related totreatment with T3. All macroscopic findings were consistent with agonalchanges, autolysis and or changes related to the intraperitonealinjections and/or intratracheal instillations. All microscopic findingsin the lungs were similar in intensity and frequency across treatmentgroups.

There were seven unscheduled deaths in this study: one vehicle control,four saline controls, and two liothyronine sodium injection animals. Inall cases the animals died shortly after intratracheal injection or werefound dead post dose. Macroscopic and microscopic findings in allanimals were consistent with autolysis and/or agonal change, and thecause of death in the liothyronine sodium injection animals was likelyrelated to the instillation procedure and not test article related.There were no macroscopic or microscopic findings in any of the endorgans examined that were determined to be related to treatment withliothyronine sodium injection.

T3 was successfully quantified for all of the samples submitted. Allreported values were within the limits of quantification for the assay(10 pg/mL-460 pg/mL). The measurable values are shown in FIG. 5 (mean±standard error). The greater serum T3 concentrations in females versusmales is likely due to the somewhat lower body weights for the females,and the finding in preliminary studies that females on average have alarger lung weight/body weight ratio than do males; the relativelylarger lung surface area allowing for greater absorption of T3 into thesystemic circulation.

Thus, T3 was administered into the lungs by direct instillation withminimal test-material-related adverse events or complications. Seventoxicity phase and zero toxicokinetic phase animals died followingdosing procedures.

No adverse T3 related clinical observations were observed during thecourse of the study. Female rats in the toxicity phase that wereadministered liothyronine sodium did not regain body weight at the samerate as males or as in control groups. The changes in clinical pathologynoted between the T3 group and control groups are considered clinicallyinsignificant and not directly related to the T3.

Peak levels of T3 were observed at the one hour time point for males andthe two hour timepoint for females. The values for the males wereconsistently lower than the corresponding time points in the females.All animals, regardless of body weight, received the same dose of T3. Onaverage, females had lower body weights than males and thereforereceived a slightly higher calculated dose per body weight and aslightly higher dose per calculated wet lung weight than males.

Administration of the T3, normal saline, or T3 vehicle via intratrachealinstillation in a rat model evaluated after five days of administrationresulted in no unscheduled deaths that could be attributed to the testor control articles, no differences in the lung weights, and nomacroscopic or microscopic findings considered to be related totreatment with T3. Thus, all test materials were successfully dosed withno adverse events or clinically significant test material relatedcomplications.

In preliminary, dose-finding studies, there were no acute effects onphysiologic parameters (e.g. respiratory rate, O₂ saturation, or heartrate) following a single intravenous delivery, or after five days ofintratracheal delivery of 3.0 μg neutral pH T3. Also, when 3.0 μg T3 wasgiven intratracheally, the T3 C_(max) was ˜ 1/17^(th) and the AUC˜⅕^(th) of what was seen following intravenous administration of 3.0 μgT3 (data not shown).

The maximum feasible dose used in this study (on a μg of T3 per lungweight basis) is 300-fold higher than the starting dose for a proposedfirst-in-human clinical trial, providing a significant safety margin.

In the preceding description and following claims, the term “and/or”means one or all of the listed elements or a combination of any two ormore of the listed elements; the terms “comprises,” “comprising,” andvariations thereof are to be construed as open ended—i.e., additionalelements or steps are optional and may or may not be present; unlessotherwise specified, “a,” “an,” “the,” and “at least one” are usedinterchangeably and mean one or more than one; and the recitations ofnumerical ranges by endpoints include all numbers subsumed within thatrange (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

In the preceding description, particular embodiments may be described inisolation for clarity. Unless otherwise expressly specified that thefeatures of a particular embodiment are incompatible with the featuresof another embodiment, certain embodiments can include a combination ofcompatible features described herein in connection with one or moreembodiments.

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted simultaneously.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example 1

Human Lung Tissue Procurement

Post-mortem lung tissue was obtained from consecutive adult patients(male and female) with a clinical diagnosis of ARDS. Autopsies wereauthorized by the Institutional Review Board and performed after familyconsent, from December 2008 through October 2009. The diagnosis of ARDSwas based on the following criteria: P_(a)O₂/FIO₂<200 mmHg, wedge <18mmHg or CVP<12 mmHg, and CXR with bilateral patchy infiltrates asdefined by the American European Consensus Conference. ARDS resultedfrom a variety of etiologies including pneumonia (viral or bacterial),sepsis, trauma and post-surgical lung injury (Table 1). Consecutiveadult patients dying of non-pulmonary causes and undergoing autopsy bythe Medical Examiner were used as controls (e.g., alcohol overdose,hypothermia, myocardial infarction, and motor vehicle trauma (Table 1).

TABLE 1 ARDS patients and normal controls Free T3 ARDS Patients ARDSEtiology (pg/mL) 23-year-old male H1N1 influenza pneumonia 1.244-year-old male H1N1 influenza pneumonia 46-year-old female MRSApneumonia/sepsis 64-year-old male PCP/CMV pneumonia 80-year-old malePneumococcal Pneumonia/sepsis 40-year-old male Cholecystitis/sepsis63-year-old female Post-CABG surgery 1.5 65-year-old female Motorvehicle trauma Free T3 Normal Controls Cause of Death (pg/mL)23-year-old male Alcohol overdose 63-year-old male Hypothermia54-year-old male Acute Myocardial infarction 3.8 48-year-old femaleMotor vehicle trauma MRSA: Methicillin-resistant Staphylococcus aureus;PCP: Pneumocystis carnii (jiroveci) pneumonia; CMV: Cytomegalovirus;CABG: Coronary artery bypass graft.

The lung samples were procured within four to twelve hours after death.Tissue samples were dissected from the peripheral/sub-pleural parenchymaof the anterior lung fields, sliced into 2-cm×2-cm pieces, flash frozenin liquid nitrogen, and stored at −80° C. for future assays or fixed informalin and embedded for histological and immunochemical analysis.Staff pathologists (Department of Pathology and Laboratory Medicine,Essentia Health-St. Mary's Medical Center and Duluth Clinic, Duluth,Minn.) assigned a histologic diagnosis to each set of tissue. Lungsamples demonstrating diffuse alveolar damage (DAD), includinghypercellularity, and hyaline membrane/fibrin deposition, were used asstudy tissues. Lung samples from patients dying of non-pulmonary causesand demonstrating normal lung histologic architecture were used ascontrol tissues. All samples with equivocal histology were excluded.

Human Lung Deiodinase III (D3) Immunohistochemistry

Immunohistochemistry for detection of D3 was performed using a primaryrabbit anti-deiodinase 3 antibody (1:100; gift of Domenico Salvatore, M.D., Ph.D., University of Naples Federico II, Naples, Italy), and abiotinylated goat anti rabbit secondary antibody followed by an avidinbiotin complex (Vector Laboratories, Inc., Burlingame, Calif.).Diaminobenzidine (DAB) was used as the chromogen. The following protocolwas used: Slides were deparaffinized in xylene and endogenous peroxidaseactivity was blocked with 0.3% hydrogen peroxide in methanol. The slideswere rehydrated and treated with trypsin for 30 minutes at 90° C. Aftercooling, the sections were blocked with 10% normal goat serum inPBS+0.1% Tween-20 for 30 minutes. The anti-D3 antibody (1:100) was addedfor one hour at room temperature followed by washing in PBS andincubation with the secondary biotinylated goat anti-rabbit IgG antibodyfor 60 minutes at room temperature. Avidin Biotin Complex (VectorLaboratories, Inc., Burlingame, Calif.) was incubated with the tissuefor 30 minutes followed by development of diaminobenzidine until thedesired staining intensity was reached. The slides were counterstainedfor one minute with hematoxylin, dehydrated and examined. All tissue wasidentically processed with equal exposure time. Examination andphotography was performed using a light microscope (DMRB, LeicaMicrosystems GmbH, Wetzlar, Germany).

D3 Enzymatic Activity

Frozen lung tissue samples were thawed and sonicated in 0.1 M phosphateand 1 mM EDTA at pH 6.9 with 10 mM dithiothreitol and 0.25 M sucrose. D3activity was assayed by HPLC using 150 μg of cellular protein, 200,000cpm of 3, 5,[125I]3′-triiodothyronine (PerkinElmer, Inc., Waltham,Mass.), 1 mM 6N-propylthiouracil (PTU), 10 mM dithiothreitol (DTT), and0-500 nM unlabeled T3 in each reaction as previously described(Simonides et al., J Clin Invest 118:975-983; 2008). Reactions werestopped by adding methanol and the products of deiodination werequantified by HPLC as previously described (Richard et al., J ClinEndocrinol Metab 83:2868-2874; 1998). D3 velocities were expressed asfmol of T3 inner-ring deiodinated per mg of sonicate protein per minute(fmol/mg/min). Samples with velocities below the detection limit of theassay were set to the minimum detectable activity (MDA) value, 0.05fmol/mg/min. The MDA was calculated statistically as three standarddeviations above background activity.

Lung Total Tissue T3 Measurement

Thyroid hormones were extracted from human lung samples weighing ˜0.5 gusing a modification of a previously-described method (Excobare et al.,Endocrinology 117:1890-1900; 1985). Tissue was homogenized in 4 mLmethanol containing 1 mM PTU (methanol-PTU) per gram tissue with arotor-stator homogenizer at 30,000 rpm for 30 seconds. To assessindividual sample percent recoveries, 100 μL of ¹²⁵I-T4 tracer (0.02pg/μL in methanol-PTU) was added to each sample. Chloroform was added atdouble the volume of methanol-PTU and samples were mixed by vortexing.The mixture was centrifuged at 2000 rpm for 15 minutes and thesupernatant liquid was transferred to a clean 50 mL tube. The remainingpellets were subjected to two additional extractions by vortexing in 5mL chloroform:methanol (2:1) per gram tissue, centrifuging at 2000 rpmfor 15 minutes, and removing and combining the supernatant with thefirst extract. To the combined extracts, 1 mL 0.05% CaCl₂ was added forevery 5 mL of extract. The mixture was vortexed and centrifuged at 2000rpm for five minutes. The upper aqueous layer, containing thyroidhormones, was transferred to a clean 50-mL tube. The lower organic layerwas re-extracted two more times with a volume of pure upper layer(chloroform:methano1:0.05% CaCl₂, 3:49:48) equal to the amount of upperlayer removed in the previous step. The combined extracted upper layerswere subjected to rotary evaporation to remove the remaining chloroformand methanol. The aqueous mixture was shell-frozen and evaporated tocomplete dryness by lyophilization. Each lyophilized sample wasdissolved in 500 μL stripped rat serum and T3 levels were measured usinga serum total T3 RIA assay kit (Siemens Medical Solutions Diagnostics;Los Angeles, Calif.), as previously described (Bastian et al.,Endocrinology 151:4055-4065; 2010).

Statistical Analysis

Statistical analysis of D3 activities and tissue T3 levels was performedusing one-way analysis of variance and Tukey's post hoc multiplecomparison test. Statistical analyses and data graphing were carried outusing the Prism (GraphPad Software, La Jolla, Calif.) software package.Data are presented as mean±SEM. An α=0.05 was chosen to definesignificant differences.

Example 2

Study Design: First In-Human Clinical Trial as Described in FDA-ApprovedInvestigative New Drug (#126204).

Informed consent is obtained within 24-hours prior to administering thestudy drug. For both the Treatment Group (Intervention) and ControlGroup (Non-Intervention), the study protocol will be started at Time 0with a 6-hour EVLWI/PVPI measurement, a 12-hour EVLWI/PVPI measurement,a 24-hour EVLWI/PVPI measurement, a 48-hour EVLWI/PVPI measurement, a72-hour EVLWI/PVPI measurement, and a 96-hour EVLWI/PVPI measurement.

Study Drug

Human ARDS patients are treated with liothyronine sodium (T3), which isa synthetic form of thyroid hormone T3. Liothyronine sodium is providedin amber-glass vials containing 10 (10 mcg/ml in 1 ml vials) ofliothyronine sodium in a sterile non-pyrogenic aqueous solution of 6.8%alcohol (by volume), 0.175 mg anhydrous citric acid, and 2.19 mgammonium hydroxide. Prior to instillation, the liothyronine sodium isadjusted to neutral pH (6-8) by adding 1.0 N HCL prior to diluting in0.9% normal saline (NS) under sterile conditions by an appropriatelytrained pharmacist.

Liothyronine sodium is formulated for administration as follows: 5 μgdose (0.5 ml liothyronine sodium+0.9% NS to 10 ml total volume); 10 μgdose (1.0 ml liothyronine sodium+0.9% NS to 10 ml total volume); 25 μgdose (2.5 ml liothyronine sodium+0.9% NS to 10 ml total volume); 50 μgdose (5.0 ml liothyronine sodium+0.9% NS to 10 ml total volume).

Treatment Group (Intervention)

50 patients receive treatment. Upon enrollment and measurement ofbaseline values, patients receive 5 μg T3 by airway instillation.Patients are monitored for 24 hours for adverse effects and changes inEVLWI. After 24 hours, if no adverse effects are seen and EVLWI and/orPVPI is unchanged, patients receive a 2× escalated dose of 10 μg T3 byairway instillation. Patients are monitored for 24 hours for adverseeffects and changes in EVLWI. and/or PVPI. At t=48 hours from first T3dose, if no adverse effects are seen and EVLWI is unchanged, patientsreceive a 2.5× escalated dose of 25 μg T3 by airway instillation.Patients are monitored for 24 hours for adverse effects and changes inEVLWI and/or PVPI. At t=72 hours from first T3 dose, if no adverseeffects are seen and EVLWI is unchanged, patients receive a 2× escalateddose of 50 μg T3 by airway instillation. A final EVLWI and PVPImeasurement is made 24-hours after final T3 dose at time=96 hours (endtime point).

Control Group (Non-Intervention)

The control group includes 18 patients. Upon enrollment and measurementof baseline values, control patients receive no research intervention.Control subjects receive standard of care. EVLWI and PVPI are measuredat Time 0 (before treatment), at six hours, 12 hours, 24 hours, 48hours, 72 hours, and 96 hours.

Primary Study Endpoints

Prior to commencing the study protocol and continuously thereafter,safety and tolerability of the airway to instilled T3 therapy areassessed. Subjects will be monitored for composite endpoints, includingpulmonary events (e.g., progressive hemoptysis; quantity ≥30 mlblood-stained sputum), cardiac events (e.g., new sustained ventriculararrhythmia (duration >30 seconds); new sustained accelerated junctionalarrhythmia (rate >80 bpm) with worsened hypotension; new sustainedatrial fibrillation with rapid ventricular response (ventricularrate >160 bpm) with worsened hypotension; or cardiac arrest (asystole orpulseless electrical activity); and/or hypertensive crisis(systolic >200, or diastolic >120, or change in MAP >20 mmHg).

Secondary Study Endpoints

To assess the efficacy of airway-instilled T3 on reducing EVLWI and/orPVPI in ARDS patients, EVLWI, PVPI, and oxygenation (arterial blood gas,ABG) are measured on subjects in both the Treatment Group and theControl Group beginning at baseline (T=0) and at six hours, 12 hours, 24hours, 48 hours, 72 hours, and 96 hours thereafter. Additional serialmeasurements include blood pressure (BP), mean arterial pressure (MAP),central venous pressure (CVP), cardiac index (CI), systemic vascularresistance index (SVRI), oxygen saturation O_(2sat), Finally, serum freeT3, free T4, and TSH are measured at each time interval.

Example 3

Test System

This study was conducted using both male and female Sprague-Dawley rats(Envigo, Huntingdon, United Kingdom). Evaluation of the safety of thetracheal route of instillation for liothyronine sodium injection inhuman clinical trials can be accomplished in this species at appropriatedose levels. Furthermore, responses to thyroid hormone in rats aresimilar to responses in humans, and the choice of the rat model is basedin large part on pharmacologic data from studies of thyroid hormone andassociated receptors and physiological responses in rat lung. TheUniversity of Minnesota is accredited by the Association for theAssessment and Accreditation of Laboratory Animal Care, International(AAALAC) and registered with the United States Department of Agricultureto conduct research in laboratory animals. Animal studies conformed toNIH guidelines (Guide for the Care and Use of Laboratory Animals. NIHpublication No. 86-23. Revised 1985). The protocol was reviewed andapproved, as applicable, by the Institutional Animal Care and UseCommittee (IACUC) at the University of Minnesota for compliance withregulations prior to study initiation or implementation of amendedactivities.

Summary of Toxicology Study Design

Details of the study design are shown in Table 1. Sixty animals (plustwo spare animals/sex/group) were anesthetized and dosed viaintratracheal instillation of test or control materials for fiveconsecutive days. On the day after the last dose a terminal bloodcollection was performed for clinical pathology, after which animalswere euthanized and a gross examination of all organs was performed by aboard certified veterinary pathologist. Select tissues were collectedfor histopathology. Twenty-four animals in the toxicokinetic (TK) phasewere anesthetized and dosed with a single intratracheal instillation ofT3. Terminal blood collection was performed at two designated timepoints per animal up to 24 hours after administration for toxicokineticevaluation. TK animals were euthanized without further evaluation afterthe final blood collection. All animals were acclimated for a minimum ofseven days prior to the dosing procedure. Animals underwent baselineobservations and examinations prior to the initiation of the study, andclinical observations and body weight monitoring were performedthroughout the in-life portion of the study. All animals received thesame dose volume (0.3 mL) of either test or control materials. Thismaximum feasible dose (MFD) is constrained by the maximum volume thatcan be safely and reproducibly (over five days) instilled into lungs ofrats weighing 250 g to 350 g, which was determined in preliminarytoxicology studies to be 0.3 mL. Actual doses delivered are reported asboth μg/kg body weight and μg/gm wet lung weight. The toxicity phaseanimals were 72-135 days of age at the time of initial dosing. Malesweighed between 256.52 g and 307.50 g with a mean±standard deviation of286.74±11.77 g, and females weighed between 250.46 g and 299.01 g with amean±standard deviation of 260.64±10.34 g. The TK phase animals were68-144 days of age at the time of initial dosing. Males weighed between261.23 g and 316.19 g; females weighed between 250.06 g and 280.32 g.

TABLE 1 Study Design Toxicokinetic Toxicity Phase (TK) Phase Number ofNumber of Toxicity Animals TK Animals Dose Group M F M F Volume 1 T3Vehicle 10 + 2^(a) 10 + 2^(a) 0 0 300 μL (Control) 2 Normal Saline 10 +2^(a) 10 + 4^(a) 0 0 (Control) 3 T3 (Test 10 + 2^(a) 10 + 2^(a)  12 +2^(b) 12 + 2^(b) Article) Totals 30 + 6  30 + 8  12 + 2 12 + 2   60 + 1424 + 4 Dosing *Once daily Single Dose Frequency & for 5 days DurationScheduled 1 day post Day 1 Termination final dose TK Blood NA Atdesignated Collection time points Histology Yes NA F = Females, M =Males, NA = Not Applicable ^(a)Use of Spares-Two spare animals of eachsex/toxicity group were dosed with the animals from each group so thatthey were available for replacement within the similar timeframe. Anadditional two females were dosed in Group 2 due to early deathsexperienced due to non-test material-related issues. The spare animalsunderwent terminal clinical pathology and gross necropsy evaluations.^(b)Use of Spares-Four (4) additional unused spares were released fromstudy at the direction of the Study Director.Test and Control Materials

T3 used for this study was liothyronine sodium injection (X-GENPharmaceuticals, Inc., Horseheads, N.Y.) supplied in 1.0 mL amber glassvials at a concentration of 10 μg in 1.0 mL. Each mL of liothyroninesodium injection contains, in sterile, non-pyrogenic USP grade water,liothyronine sodium equivalent to 10 μg of liothyronine (T3), 6.8%alcohol by volume, 0.175 mg anhydrous citric acid and 2.19 mg ammonia(as ammonium hydroxide). In preliminary studies it was determined thatrats do not tolerate intratracheal instillation of liothyronine sodiumat a pH of >10.0 as it is supplied commercially. Therefore, theliothyronine sodium and the vehicle were adjusted to neutral pH(6.0-8.0) with sterile 1.0 N HCl (Sigma-Aldrich, St. Louis, Mo.) addedaseptically in a biosafety cabinet prior to intratracheal instillationinto animals. Using aseptic technique, vials of liothyronine sodium wereopened and the entire contents were transferred into sterile 1.5 mLEppendorf tubes. 80-90 μL of 1.0 N HCl was added to the tube and gentlyvortexed, and the pH was measured using pH test strips (pH 4.5 to 10.0,Ricca Chemical Co., Arlington, Tex.). Additional HCl was titrated ingradually, as needed, until the pH was in the desired range. There is avolume increase of approximately 10% after adjusting the pH to neutralwith 1.0 N HCl, resulting in a final concentration of liothyroninesodium of approximately 2.73 μg T3/300 μL (9.17 μg/ml). This solutionwas stored at 4° C. and used for up to 26 hours after pH adjustmentprocedure. The vehicle control solution was prepared in sterile,non-pyrogenic USP grade water (USP/EP Purified, Ricca Chemical Co.,Arlington, Tex.) and contained, per mL of solution, 6.8% ethanol (DeconLaboratories, Inc., King of Prussia, Pa.) by volume, 0.175 mg anhydrouscitric acid (Sigma-Aldrich, St. Louis, Mo.) and 2.19 mg ammonia (J. T.Baker Chemical Co., Avantor Performance Materials LLC, Radnor, Pa.),ammonia solution, strong 27.0-30.0%, N. F.-F. C. C.). The vehicle wasalso adjusted to neutral pH (6.0-8.0) with 1.0 N HCl as described above.Using aseptic technique the vehicle was filter sterilized and aliquotedinto 21 sterile disposable tubes (5 mL each) and stored at 4° C., and anew tube was opened for each day of use. Normal Saline (0.9% SodiumChloride Injection USP, B. Braun Medical, Inc., Bethlehem, Pa.), wasstored at room temperature. A new package was opened for each day ofuse.

In-Life Animal Care

Upon arrival animals were visually examined by trained staff andweighed, counted, sexed, and appropriately separated into housing boxes.Each animal received a metal ear tag containing an individual identifierprior to initial dosing. Animals were housed in AAALAC accredited pensunder sanitary conditions and were socially housed to provide enrichmentand companionship. The temperature and humidity of the housing area wasmonitored a minimum of once daily. Animals were acclimated for a minimumof seven days prior to dosing initiation. Preconditioning was allowedduring this period to acclimate the animals to the handling they wouldexperience during weighing, examinations and dosing procedures. Allanimals were given food (TEKLAD, Envigo, Huntingdon, United Kingdom) andpotable tap water ad libitum. Animals were not fasted for procedures.Veterinary care was available throughout the course of the study.Observations on general health, including animal activity, appearance,food and water intake, mortality/moribundity and other endpoints (Table2) were performed and recorded at least once daily from the time ofenrollment on study until euthanasia by a trained technician. AVeterinarian was notified of abnormalities in activity or appearance. Toprevent bias with regard to observations, health concerns or treatments,veterinary and general animal care personnel were not informed of dosegroup distribution.

TABLE 2 In-life Observations/Assessments & Health Monitoring ActivityFrequency Handling & Restraint SID beginning within 2 days after arrivalPre-Conditioning Body Weight (grams) Within 1 days prior to and/or onthe day of initial dose. Minimally 2-3 times/week during dosing Day ofterm Cage Side Observations Minimally SID beginning within 3 daysMortality/Alive or Dead prior to initial dose. Defecation Day of termUrination Behavior/vocalization Posture/Attitude/Activity Food availableWater available, bottle intact Bedding sufficient Detailed ClinicalObservations At least once prior to initial dosing Posture/Attitude Atleast once after initial dose Integument/Hair Day of Term Eyes Ears NoseRespiratory Musculoskeletal Urogenital Gastrointestinal NeurologicalDosing Procedure

Test and control materials were drawn into dosing syringes using aseptictechnique. Using a 18G needle, 0.5 mL of air was drawn into a 1 ccsyringe followed by 0.3 mL (300 μL) of the solutions. Animals wereanesthetized with a combination of ketamine, 40 mg/kg to 200 mg/kg, andxylazine, 1 mg/kg to 7 mg/kg intraperitoneally (IP), to effect. The dosewas adjusted daily, as needed, based on individual animal response andrecovery. Depth of anesthesia was evaluated by toe pinch, and eyelubricant was applied to the eyes. An upright, inclined stand was usedto support the animals in the desired position during the dosingprocedure by suspending the animals from a soft, non-latex rubber bandat the top of the stand by their front incisors. Up to 20 μL of 2%lidocaine was applied topically to the back of the throat using a bluntgavage needle prior to intubation with a tracheal catheter to minimizelaryngeal spasms and facilitate tracheal placement. The animals wereremoved from the stand and positioned in prone position while thelidocaine took effect.

After allowing adequate time for lidocaine to take effect, the animalswere again suspended on the apparatus, and a catheter (INTRAMEDIC 1.19mm inner diameter, 1.70 mm outer diameter, Thermo Fisher Scientific,Waltham, Mass.) was inserted into the trachea by first visualizing thelarynx through the oral cavity with the aid of an external light sourcedirected at the throat. Holding the tongue aside with blunt forceps andgauze moistened with water helped with visualization of the airway. Thecatheter was advanced into the trachea to a pre-determined depthapproximately 1.0 cm short of the branch point of the major bronchi(measured on a cadaver animal with the trachea and bronchi exposed).Catheter placement in the airway was verified by the fogging of a dentalmirror placed at the opening of the catheter. The needle on the dosingsyringe was then inserted into the catheter, and the test material andbolus of air was rapidly delivered in a one-to-two second interval. Theair bolus administered after the test material facilitatedadministration of the fluid into the lower airways and ensured thatfluid was not retained in the trachea or major bronchi, as confirmed inpreliminary experiments using a dye solution. The tracheal catheter wasremoved from the airway and the animal gently removed from the supportapparatus. The animal was placed in a prone position on a heating padwith the chest elevated for a minimum of two minutes after instillation.After two minutes, the animal was placed flat on a heating pad untilfully recovered.

Terminal Euthanasia Procedures

Blood Collection for Clinical Pathology (Hematology and ClinicalChemistry)

Blood samples from the toxicity phase animals for clinical pathologywere collected one day after the final (fifth) intratracheal dose.Animals were anesthetized with isoflurane 2-5% and oxygen 1-1.5 L/min byinhalation anesthesia via nose cone as needed. For hematology, ≥0.5 mLwhole blood was collected via the orbital sinus through plain or coatedmicrohematocrit capillary tubes into K₂EDTA collection tubes (BDBiosciences, Thermo Fisher Scientific, Waltham, Mass.) containing anadditional 30 μL of 2% EDTA solution (Sigma-Aldrich, St. Louis, Mo.),and kept at 4° C. until same day analysis. For serum chemistry, ≥0.75 mLwhole blood was collected via the orbital sinus through uncoatedcapillary tubes into red top serum microtubes (Sarstedt AG & Co. K G,Nümbrecht, Germany). For serum collection, tubes were maintained at roomtemperature for 30 to 60 minutes after collection and then centrifugedat 10,000×g for five minutes at 4° C. The resultant serum was separatedand stored at ≤−70° C. if analysis was to occur the following day orkept at 4° C. for same day analysis. All samples were sent to theUniversity of Minnesota-Veterinary Medical Center (VMC) clinicalpathology laboratory for analysis. Parameters evaluated for hematologyare provided in Table 3. Parameters evaluated for clinical chemistry areprovided in Table 4. Following blood collections animals were euthanizedwith EUTHASOL (Virbac Corp., Fort Worth, Tex.) ≥86 mg/kg IP to effectprior to necropsy. Assessment of the clinical pathology values wasperformed by Jill Schappa Faustich, DVM, DACVP, University of Minnesota.

TABLE 3 Hematology Red Blood Cell Count (RBC) Hemoglobin Concentration(HGB) Free Plasma HGB Hematocrit (HCT) Mean Corpuscular Volume (MCV)Mean Corpuscular Hemoglobin (MCH) Mean Corpuscular HemoglobinConcentration (MCHC) Reticulocyte Count (Retic Absolute And Relative)Platelet Count (Plt & PCT Absolute And Relative) Mean Platelet Volume(MPV) Platelet Distribution Width (PDW) Red Cell Distribution Width(RDW) White Blood Cell Count (WBC) Neutrophil Seg Count (NEUT AbsoluteAnd Relative) Neutrophil Band Count (BAND Absolute And Relative)Lymphocyte Count (LYMPH Absolute And Relative) Monocyte Count (MONOAbsolute And Relative) Eosinophil Count (EOS Absolute And Relative)Basophil Count (BASO Absolute And Relative) Mast Cells (Absolute AndRelative) Unclassified Cell Count (Absolute And Relative)

TABLE 4 Serum Chemistry Blood Urea Nitrogen (BUN) Creatinine (Creat)Calcium (Ca) Phosphorous (Phos) Magnesium (Mg) Protein (TP) Albumin(Alb) Globulin (Glob) Alb/Glob ratio^(a) Sodium (Na) Chloride (Cl)Potassium (K) Bicarbonate (HCO3) Osmolality (Osmol) Anion Gap (An Gap)Bilirubin, Total (T.Bili) Alkaline Phosphatase (ALP) Gamma-GlutamylTransferase(GGT) Alanine Transferase (ALT) Aspartate Transferase (AST)Creatine Kinase (CK) Glucose (Gluc) Cholesterol (Chol) Amylase LipemiaIcterus Hemolysis (LIH)Toxicokinetics (TK)Blood Collection

For toxicokinetic experiments, rats were anesthetized with combinationof ketamine 40 mg/kg to 200 mg/kg and xylazine 1 mg/kg to 7 mg/kg,intraperitoneally (IP), to effect for dosing procedures, and dosedintratracheally with liothyronine sodium injection as previouslydescribed. The details of the TK sample collection protocol are providedin Table 5 and Table 6. Depending on the duration of time between dosingand the first or second blood collection time points, animals either hadblood collected while still anesthetized under the injectableanesthetics, or if recovered, they were anesthetized with Isoflurane2-5% and oxygen 1-1.5 L/minute by inhalation anesthesia via nose cone,as needed, to maintain adequate anesthesia depth (assessed by toepinch). Topical proparicaine anesthetic ophthalmic solution was appliedto each eye prior to performing the first blood collection and allowedtime to take effect. Collection of serum samples for TK analysis was asdescribed for serum chemistry samples above, and samples were stored at≤−70° C. until assayed. Animals were euthanized with EUTHASOL (VirbacCorp., Fort Worth, Tex.) ≥86 mg/kg IP to effect following the finalblood collection.

TABLE 5 Toxicokinetic Blood Collections Sample Collection Time PointsTotal Number Number of animals M/F per Occasion (Target Time Post Dose)TK of Males/ Two time points per animal Group Females T0 15 min 30 min 1hr 2 hr 4 hr 6 hr 24 hr Test 12/12 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3Article

TABLE 6 Toxicokinetic Animal Distribution per Timepoint T0 15 min 30 min1 hr 2 hr 4 hr 6 hr 24 hr Male X X Male X X Male X X Male X X Female X XFemale X X Female X X Female X X Three animals of each sex/timepointThree Animals of Each Sex/TimepointBioanalytical Procedure

For assessment of serum T₃ levels samples were sent to the FairviewUniversity of Minnesota Medical Center East Bank Diagnostic Laboratoryfor analysis, a clinical laboratory certified by CLIA and CAP. Prior tosending serum samples to the analytical lab each sample was diluted 1:4or 1:8 in normal (0.9%) saline. These dilutions, determined inpreliminary studies, ensured that sample total T3 concentrations wouldfall within assay range (10 pg/mL to 460 pg/mL). Samples were analyzedby a chemiluminescence assay for total triiodothyronine (T3).

TK Analysis

An evaluation of the TK analysis was performed by Dick Brundage, PhD,University of Minnesota. Toxicokinetic parameters were estimated usingPhoenix 64 WinNonlin pharmacokinetic software version 7.0. (PharsightCorp., Mountain View, Calif.). A non-compartmental (NCA) approachconsistent with the route of administration was used for parameterestimation. All parameters (Table 7) were generated from mean T3concentrations in serum from all timepoints unless otherwise stated.Whenever possible, mean concentrations were derived from threeanimals/gender/time point. Parameters were estimated using samplingtimes relative to the start of each dose administration. The raw datawas converted to ng/ml of serum by dividing the pg/dl values by 100 andthen multiplying by the dilution factor for that sample, either 4 or 8.Values below the limit of quantification were calculated as 0.

TABLE 7 TK Parameters Estimated Parameter Description of parameterC_(max) The maximum observed arithmetic mean concentration of T₃measured after dosing. C_(max)/D The C_(max) divided by the doseadministered. T_(max) The time after dosing at which the maximumobserved arithmetic mean concentration of T₃ was observed. AUC_((0-t))The area under the T₃ arithmetic mean concentration versus time curvefrom time zero the time after dosing at which the last quantifiableconcentration of the drug was observed estimated by the linear orlinear/log trapezoidal method. AUC_((0-t))/D The AUC_((0-t)) divided bythe dose administered. When data permitted, the slope of the terminalelimination phase of each arithmetic mean concentration versus timecurve was determined by log-linear regression, and the followingadditional parameters were estimated: Additional Parameters EstimatedParameter Description of parameter T½ The apparent terminal eliminationhalf-life. AUC_((0-inf)) The area under the arithmetic meanconcentration versus time curve from time zero to infinity.AUC_((0-inf))/D AUC_((0-inf)) divided by the dose administered. CLClearance: the apparent volume of plasma cleared of T₃ per unit timefollowing intravenous dosing. V_(d) The apparent volume of distributionof T₃, determined from the terminal elimination phase followingintravenous dosing.

Calculation of arithmetic means and standard deviations for the matrixconcentration data was performed/replicated in EXCEL (Microsoft, Corp.,Redmond, Wash.) for reporting purposes. In addition to parameterestimates from mean concentration vs. time curves, the standard error ofthe AUC_((0-t)) and C_(max) by dose group, day, and gender (asappropriate) were generated using WINNONLIN (Cetara USA, Inc.,Princeton, N.J.).

C_(max) and T_(max) were obtained by inspection of the data. Sincemeasurable endogenous compound is present based on the observedconcentration at time zero, a baseline subtraction was performed. Usingthe mean concentration data, the concentration at time zero wassubtracted from the remaining concentrations for male and femaleanimals. The area-under-the-curve (AUC) of the baseline subtractedconcentrations was calculated using the linear trapezoidal rule. Sincethe 24-hour concentration in both male and female animals hadapproximately returned to the baseline (pre-dose) concentration, theseobservations were ignored in calculations for the AUC and half-life. Theterminal elimination half-life was calculated from the last threeobservations at 2 hours, 4 hours, and 6 hours. WinNonlin NCA performslinear regression on the logs of the concentrations. The Uniformweighting scheme was selected. The default regression algorithm for NCAwill not use C_(max) in the calculation of half-life, even if it appearsto be part of the log-linear profile, nor will it provide any half-lifebased on only two observations. The default regression for the maleanimals was used. However, for the female animals, the concentration attime two hours was also the C_(max) value. Since it appeared to fall onthe regression line of all three concentrations (adjusted Rsquared=1.0), it was included in the calculation of the half-life.Parameters were evaluated as appropriate at the discretion of theevaluator. Results are provided as individual values, and includegraphing of mean and standard error using EXCEL (Microsoft, Corp.,Redmond, Wash.) and WINNONLIN (Cetara USA, Inc., Princeton, N.J.) perappropriate groups when possible.

Necropsy Procedures

Gross Pathology

Toxicity Phase animals that were euthanized at scheduled termination orthat were found dead or euthanized prior to scheduled termination, weresubjected to an extensive necropsy performed by a board certifiedveterinary pathologist. The necropsy included an examination of theanimal carcass and musculoskeletal system, external surfaces and all ofits orifices, and cervical, thoracic, abdominal and pelvic regions,cavities and contents. Eyes were not examined due to terminal orbitalblood collection methods.

Histopathology

The primary target tissues assessed in this study for histopathalogicchanges included the lungs, the trachea-bronchi branch point and thetracheobronchial lymph nodes. The intact heart-lung pluck including alltarget tissues noted above was removed from the animal intact. Theheart-lung pluck was weighed, photographed and the lungs were thenperfusion inflated via the trachea with 10% neutral buffered formalin(NBF). For inflation, an 18 g butterfly catheter connected to areservoir of 10% NBF was inserted into the trachea and the lungsinflated for two minutes at a constant pressure of ˜20-25 cm, afterwhich the trachea was tied off with suture to maintain inflation of thelungs during fixation. The entire heart lung pluck was then immersionfixed in 10% NBF. Prior to further processing for histology, the heart,trachea and any other adherent tissues were removed from the lungs andweighed. This weight, when subtracted from the weight of the heart-lungpluck taken at necropsy, provided the wet lung weight used in subsequentcalculations of actual dose delivered. Non-target tissues including thebrain, heart, liver, spleen, pancreas, kidneys and adrenal glands wereevaluated for gross lesions. The non-target organs were collected wholewith the exception of the liver, in which a representative specimen wascollected from the anterior right lobe, and were stored in 10% NBF forpotential future analysis. Histological processing and evaluations wereperformed by Dr. Joan Wicks, DVM, PhD, DACVP, Alizée Pathology, LLC,Thurmont, Md.

Dose Administration

All doses were administered via intratracheal instillation at themaximum volume that could be safely and reproducibly delivered daily forfive consecutive days, determined in preliminary studies to be 0.3 mL(300 μl) for rats weighing 250-350 grams. There were no apparentcomplications with the administration of the materials with theexception of one instance of 20-50 μl of vehicle control article comingout of the top of the dosing syringe during Dose 3 administration to LRT633 (T3 vehicle group).

The calculated dose of T3 administered based on body weight on theinitial day of administration (Day 1) and based on calculated wet lungweights are detailed in Table 8.

TABLE 8 Calculated T3 Dose. Toxicity Phase Group 3 Group 3 CalculatedDose Group 3 All Males Females μg T3/g Wet Lung Average  1.57 (0.61)1.50 (0.15)  1.63 (0.14) Weight (SD) μg T3/kg Body Average 10.00 (0.64)9.45 (0.33) 10.55 (0.25) Weight (SD) 1.0 ml T3 (10 μg/ml) diluted with~100 μl 1.0N HCl to pH, 10 μg in 1.10 ml = 2.73 μg in 300 μl doseToxicokinetics (TK)

Liothyronine sodium (T3) was successfully quantified for all of thesamples submitted. All reported values were within the limits ofquantification for the assay (10 pg/mL-460 pg/mL).

The measurable values are listed in Table 9 and are graphed in FIG. 5(mean±standard error). TK analysis was performed on diluted samples fromall 24 animals that received a single T3 dose (Table 10).

TABLE 9 T3 Detected in Serum in Single Dose TK Study T₃ (ng/mL) Meansand Standard Errors of Mean (SEM) 0 15 min 30 min 1 hr 2 hr 4 hr 6 hr 24hr Male 1.16 2.24 3.48 8.44 6.72 6.08 3.96 0.76 1.28 2.52 3.76 6.84 6.924.52 3.24 0.80 1.20 2.44 3.36 8.16 7.28 5.36 3.64 0.72 Mean 1.21 2.403.53 7.81 6.97 5.32 3.61 0.76 SEM 0.04 0.08 0.12 0.49 0.16 0.45 0.210.02 Female 1.44 2.56 4.92 15.60 17.68 12.32 7.52 1.84 0.88 3.20 5.1616.08 18.32 11.48 6.92 1.24 1.04 3.24 7.44 13.68 16.96 10.00 7.64 1.60Mean 1.12 3.00 5.84 15.12 17.65 11.27 7.36 1.56 SEM 0.17 0.22 0.80 0.730.39 0.68 0.22 0.17

TABLE 10 Noncompartmental analysis of TK samples Cmax Cmax_D TmaxHL_Lambda_z AUClast Sex (ng/mL) (ng/mL/ug) (hr) (hr) (hr*ng/mL) F 16.536.12 2.00 2.85 64.08 M 6.60 2.44 1.00 3.17 25.38 AUCINF_obs AUCINF_D_obsCl_F_obs Vz_F_obs Sex (hr*ng/mL) (hr*ng/mL/ug) (mL/hr) (mL) F 89.7033.22 30.10 123.60 M 36.34 13.46 74.29 339.44

Example 4

Cell Culture and Hyperoxia Exposure

The adult rat AT2 cell line RLE-6TN (ATCC, Manassa, Va.) was cultured inDMEM/F12 medium with 10% FBS and in a 95% air, 5% CO₂ environment untilthey reached ˜50% confluence, then the cells were exposed to 95% O₂, 5%CO₂ in the presence or absence of T3 in DMEM/F12 with 2% stripped FBSfor specified time periods. At the end of the hyperoxia exposure period,the viable cells were counted by trypan blue dye exclusion.

Nuclear Extraction

Nuclei were extracted with NE-PER nuclear and cytoplasmic extractionreagents kit (Thermo Fisher Scientific, Inc., Waltham, Mass.) followingthe manufacturer's instruction.

Cell Lysis and Western Blot

The cells were lysed in lysis buffer containing 20 mM Tris·HCl (pH 7.5),150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (vol/vol) Triton X-100 withprotease inhibitors (1 mM PMSF, 2 μg/ml pepstatin, and 10 μg/ml each ofaprotinin and leupeptin), and phosphatase inhibitors (2.5 mM sodiumpyrophosphate, 1 mM β-glycerophosphate, and 1 mM Na₃VO₄). The lysate wasdrawn 10 times through a 25-gauge needle on ice for further lysis andthen was centrifuged at 13,000 rpm for 15 minutes at 4° C. Thesupernatant was collected, and the protein concentrations weredetermined by use of the BCA protein assay kit (Sigma-Aldrich, St.Louis, Mo.). Immediately after this step, equal amounts of protein weresubjected to Western blotting analysis.

Nuclear Extraction

Nuclei were extracted with NE-PER nuclear and cytoplasmic extractionreagents kit (Thermo Fisher Scientific, Inc., Waltham, Mass.) followingthe manufacturer's instruction.

Statistics

All data are expressed as means±SD of a minimum of three or moreindependent experiments, unless otherwise noted. In most experiments,individual data points within an experiment represent the mean of atleast two replicates. Comparisons involving three or more groups wereanalyzed by ANOVA and post hoc pairwise comparisons. Differences betweenmeans were considered significant at P<0.05.

Example 5

Experimental Design

All experimental protocols for animal treatments were approved by theUniversity of Minnesota Institutional Animal Care and Use Committee.Specific pathogen-free (SPF) adult male Sprague Dawley rats (250 g-300g) receiving intraperitoneal (ip) injections of either saline or T3 wereexposed to normobaric hyperoxia (FI_(O2)>95%, 5 LPM) in a chamber withad libitum access to food and water at room temperature for 48 or 60hours to induce lung inflammation and injury. The room-air control ratswere kept in the University animal housing facility. Rats were injectedintraperitoneally with T3 or saline at doses and time points detailed intwo protocols summarized in Table 11. At the end of hyperoxic exposure,rats were sacrificed by intraperitoneal pentobarbital injection, and thelungs were harvested; the right lobes were allocated for histopathology,measurement of lung tissue myeloperoxidase (MPO) activity and wet-to-dryweight ratio. The left lobes underwent bronchoalveolar lavage (BAL) todetermine BAL protein concentrations and differential cell counts.

TABLE 11 Experimental protocols 0 hours 12 hours 24 hours 36 hours 48hours 60 hours Protocol 1 Control (saline) ip ip ip ip ip end T3 (12.5μg/kg) ip ip ip ip ip end Protocol 2 Control (saline) ip ip end T3 (15.0μg/kg) ip ip endWet-Dry Lung Weight Ratios

A portion of the right lung was rinsed briefly in PBS, blotted, and thenweighed to obtain the “wet” weight. Lungs then were dried in an oven at80° C. for seven days to obtain the “dry” weight.

Lung Lavage Analyses

Bronchoalveolar lavage (BAL) of the left lung was performed using amodification of a method previously described (Pace et al., Exp Lung Res35:380-398, 2009). Briefly, 4 mls of ice-chilled 1× PBS (pH 7.4) wereinstilled into the left lung, withdrawn, and re-instilled two subsequenttimes prior to analysis of the lavage fluid. The retrieved BAL fluid wascentrifuged at 1500 rpm for 10 minutes to remove cells and debris. Thecell pellet was resuspended in 1 ml of 1× PBS (pH 7.4) and total cellnumber was counted using a hemocytometer. BAL cytospin preparations werestained using the Hema3 stain kit (Thermo Fisher Scientific, Inc.,Waltham, Mass.) to identify the nucleated cells. The proteinconcentration was determined on the supernatants of BAL fluid using astandard BCA assay (Sigma-Aldrich, St. Louis, Mo.).

Myeloperoxidase (MPO) Assay

To quantify the neutrophil activity in the lung, MPO activity wasassayed as previously described (Abraham et al., J Immunol165:2950-2954, 2000). Lung tissues without prior lavage were frozen inliquid nitrogen, weighed, and stored at −86° C. The lungs werehomogenized for 30 seconds in 1.5 ml 20 mM potassium phosphate, pH 7.4,and centrifuged at 4° C. for 30 minutes at 40,000×g. The pellet wasresuspended in 1.5 ml 50 mM potassium phosphate, pH 6.0, containing 0.5%hexadecyltrimethylammonium bromide, sonicated for 90 seconds, incubatedat 60° C. for two hours, and centrifuged at 14,000 rpm for 30 minutes at4° C. The supernatant was assayed for peroxidase activity corrected tolung weight. MPO was expressed as activity per gram of lung tissue.

Histochemistry

Lung tissue was removed and inflation fixed at 20 cm water pressure in4% paraformaldehyde, paraffin embedded, cut as 5 micron sections andmounted onto poly-L-lysine slides. Sections were deparaffinized inxylene, rehydrated through a graded alcohol series in methanol, andplaced in a 98° C. water bath for 30 minutes in citrate buffer (pH 6.0)for antigen retrieval. After quenching with 0.3% hydrogen peroxide inPBS, sections were incubated in normal serum for 30 minutes and for 15minutes each with Avidin/Biotin Blocking Kit (Vector Laboratories, Inc.,Burlingame, Calif.). After overnight incubation with MyeloperoxidaseAb-1 (Thermo Fisher Scientific, Inc., Fremont, Calif.) at 4° C.,Biotinylated goat anti-rabbit IgG (1:500) and RTU Streptavidin (VectorLaboratories, Inc., Burlingame, Calif.) were applied sequentially for 30minutes and 3,3′-diaminobenzidine was used as a peroxidase substrate.Sections were counterstained with hematoxylin. Image analysis andphotography used a Leica Leitz DMRB microscope.

Serum T3 Measurements

Blood samples were collected at the end of 60 hours of hyperoxia andwere centrifuged at 13,000 rpm for 30 minutes at 4° C. Supernatant wasstored at −20° C. Serum total T3 concentrations were measured withcommercial RIA kits (Siemens Medical Solutions Diagnostics, Los Angeles,Calif.) as previously described (Bastian et al., Endocrinology151:4055-4065, 2010).

Statistical Analysis

Values were expressed as means±SD of a minimum of three experiments.Comparisons involving three or more groups were analyzed by ANOVA andpost hoc pairwise comparisons. Differences between means were consideredsignificant at p<0.05, adjusted for the number of comparisons.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference in their entirety. In theevent that any inconsistency exists between the disclosure of thepresent application and the disclosure(s) of any document incorporatedherein by reference, the disclosure of the present application shallgovern. The foregoing detailed description and examples have been givenfor clarity of understanding only. No unnecessary limitations are to beunderstood therefrom. The invention is not limited to the exact detailsshown and described, for variations obvious to one skilled in the artwill be included within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

What is claimed is:
 1. A pharmaceutical composition for administeringdirectly to the lung of a subject, the composition comprising: a salt oftriiodothyronine in an amount effective to provide a lung-delivered doseof from 100 ng to 2 mg; and a pharmaceutically acceptable buffer;wherein the composition delivered to the lung has a pH from 5.5 to 8.5and a maximum volume of no more than 0.30 ml per 1.67 g wet lung weightper dose.
 2. The pharmaceutical composition of claim 1, wherein thecomposition is aerosolized.
 3. The pharmaceutical composition of claim1, wherein the composition is nebulized.
 4. A method for treating asubject having, or at risk of having inflammation of lung tissues, themethod comprising: administering to the subject an amount of thecomposition of claim 1 effective to ameliorate lung inflammation,wherein the composition is administered directly to the pulmonary tract.5. The method of claim 4, wherein the composition is administered byintratracheal instillation.
 6. The method of claim 4, wherein thecomposition is administered by inhalation of an aerosolized formulation.7. The method of claim 4, wherein the composition is administered byinhalation of a nebulized formulation.
 8. The method of claim 4, whereinthe composition is administered prior to the subject manifesting anysymptom or clinical sign of lung inflammation.
 9. The method of claim 4,wherein the composition is administered after the subject manifests asymptom or clinical sign of lung inflammation.
 10. The method of claim4, wherein the lung inflammation is a clinical sign of acute respiratorydistress syndrome (ARDS).
 11. The method of claim 4, wherein the lunginflammation is a symptom or clinical sign of premature birth, chesttrauma, congestive heart failure, lung transplant, lung cancerradiotherapy, lung cancer chemotherapy, smoking, exposure to apollutant, hypersensitivity pneumonitis, a reactive/obstructive lungdisease, aspiration chemical pneumonitis/pneumonia, pneumonia, aninfection of the nasosinus, intratracheal, intrabronchial or alveolarairspace, a connective tissue disease, Wegener's granulomatosis, Goodpasture disease, acute eosinophilic pneumonia, chronic eosinophilicpneumonia, medication-related lung injury, cryptogenic organizingpneumonia, Churg-Strauss syndrome, congenital lung disease, orstructural lung disease.
 12. A method for treating a subject having, orat risk of having lung injury, the method comprising: administering tothe subject an amount of the composition of claim 1 effective toameliorate a symptom or clinical sign of lung injury, wherein thecomposition is administered directly to the pulmonary tract.
 13. Themethod of claim 12, wherein the composition is administered byintratracheal instillation.
 14. The method of claim 12, wherein thecomposition is administered by inhalation of an aerosolized formulation.15. The method of claim 12, wherein the composition is administered byinhalation of a nebulized formulation.
 16. The method of claim 12,wherein the composition is administered prior to the subject manifestingany symptom or clinical sign of lung injury.
 17. The method of claim 12,wherein the composition is administered after the subject manifests asymptom or clinical sign of lung injury.
 18. The method of claim 12,wherein the lung injury is a clinical sign of acute respiratory distresssyndrome (ARDS).
 19. The method of claim 12, wherein the lung injury isa symptom or clinical sign of premature birth, chest trauma, congestiveheart failure, lung transplant, lung cancer radiotherapy, lung cancerchemotherapy, smoking, exposure to a pollutant, hypersensitivitypneumonitis, a reactive/obstructive lung disease, aspiration chemicalpneumonitis/pneumonia, pneumonia, an infection of the nasosinus,intratracheal, intrabronchial or alveolar airspace, a connective tissuedisease, Wegener's granulomatosis, Good pasture disease, acuteeosinophilic pneumonia, chronic eosinophilic pneumonia,medication-related lung injury, cryptogenic organizing pneumonia,Churg-Strauss syndrome, congenital lung disease, or structural lungdisease.